Patent Publication Number: US-2016238615-A1

Title: Solid phase extraction of global peptides, glycopeptides, and glycans using chemical immobilization in a pipette tip

Description:
CROSS-REFERENCE 
     This application claims the benefit of U.S. Provisional Application No. 61/883,635, filed on Sep. 27, 2013, which application is incorporated herein by reference in its entirety. 
    
    
     FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with government support under U01CA152813, U24CA160036, P01HL107153 and R01CA112314 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. 
    
    
     BACKGROUND 
     Proteomics analysis is important for characterizing tissues or body fluids to gain biological and pathological insights. This could lead to the identification of disease-associated proteins as disease diagnostics or therapeutics. Glycoproteins modified by oligosaccharides are expressed as transmembrane proteins, extracellular proteins, or proteins secreted to body fluids, such as blood serum, which is an excellent source for diagnosis and monitoring of the presence and stage of many diseases (Wang et al., 2013; Zhang et al., 2013). As an easily accessible body fluid, human serum contains a large array of proteins that are derived from cells and tissues all over the body. Thus, the human serum proteome contains valuable information where biomarkers may be discovered for clinical use, e.g. CA125 for ovarian cancer and PSA for prostate cancer (Maggino and Gadducci, 2000; Schroder et al., 2007). It is considerably important to study protein glycosylation and the associated glycans for diagnostics and disease prognostics. Unlike other protein modifications, glycans attached to proteins are enormously complex. Development of the high-throughput method for extraction of peptides, glycopeptides, and glycans will facilitate proteomics, glycoproteomics, and glycomics analyses. 
     To analyze glycoproteins, a robust method for isolating formerly N-linked glycopeptides using solid-phase extraction of N-linked glycopeptides from glycoproteins (SPEG) has been widely used (Zhang et al., 2003). This method isolates formerly N-linked glycopeptides containing glycosylation sites for N-glycans attachments and analyzes the peptides by mass spectrometry. Human serum N-linked glycoproteome is of special interest for a number of reasons (Zhang et al, 2006; Zhou et al., 2007). First, by focusing on formerly N-linked glycopeptides, the complexity of the proteome is greatly reduced by only analyzing 1-2 N-glycosite containing peptides for each protein (Zhang et al., 2005). Second, the high abundant non-glycoproteins, e.g., albumin, which accounts for approximately 50% of proteins in human serum, are eliminated for mass spectrometry analysis. Third, glycoproteins account for most of the serum proteins that are derived from tissues where biomarkers may be identified. Fourth, aberrantly glycosylated peptides can be specifically isolated and analyzed using enrichment of glycopeptides with specific glycans (Tian et al., 2012; Li et al., 2011). 
     Numerous studies have been carried out using the SPEG method for cancer biomarker discovery in serum and other body fluid including breast, ovarian, lung and liver cancers (Boersema et al., 2013; Wu et al., 2013; Li et al., 2013; Sanda et al., 2013). The SPEG method includes coupling of glycoproteins to a solid support using hydrazide chemistry and removal of non-glycoproteins, proteolysis of captured glycoproteins to hydrazide with trypsin, removal of digested non-glycopeptides with washing, and specific release of N-glycopeptides using peptide-N-glycosidase F (PNGase F). This procedure provides a straightforward work flow with good protein/peptide identification and specificity. The procedure, however, requires a long processing time, such as four days (Zhang et al., 2003; Zhou et al., 2007), and is hard to scale up. In addition, the procedure releases the formerly N-linked glycopeptides containing N-glycosylation sites from their attached glycans and loses the information of glycans and total proteins from the samples where the glycopeptides are from. 
     SUMMARY 
     In one aspect, the presently disclosed subject matter provides a pipette tip comprising an elongate body having a proximal end adapted to connect to and be in fluid communication with an outlet of a fluid dispensing device and a distal end having an opening adapted to dispense a fluid, the elongate body further comprising a fluid path between the proximal end and the distal end, wherein the fluid path comprises: (a) a first frit proximate the distal end and a second frit proximate the proximal end, and wherein the fluid path comprises a solid phase disposed between the first frit and the second frit, the solid phase comprising: (i) a chemical moiety capable of conjugating one or more glycoproteins through one or more oxidized glycans; or (ii) an amino-reactive moiety capable of conjugating one or more amino groups of one or more proteins disposed in the fluid path between the first frit and the second frit; or (iii) other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications disposed in the fluid path between the first frit and the second frit; or (b) a monolith-bonded aldehyde-reactive chemical moiety, a monolith-bonded amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications. 
     In certain aspects, the presently disclosed subject matter provides a method for preparing a pipette tip, the method comprising: (a) providing a pipette tip comprising an elongate body having a proximal end adapted to connect to and be in fluid communication with an outlet of a fluid dispensing device and a distal end having an opening adapted to dispense a fluid; and (b) forming a fluid path between the proximal end and the distal end by one of: (i) disposing a first frit proximate the distal end of the pipette tip and disposing thereon a solid phase comprising one of a chemical moiety capable of conjugating one or more glycoproteins through one or more oxidized glycans or an amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications capable of conjugating one or more amino groups of one or more proteins, and disposing a second frit proximate the proximal end of the pipette tip; or (ii) disposing a monolith-bonded aldehyde-reactive chemical moiety or a monolith-bonded amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications between the distal end and the proximal end of the pipette tip. 
     In particular aspects, the presently disclosed subject matter provides a kit comprising at least one presently disclosed pipette tip, wherein the kit further comprises a set of instructions for using the at least one pipette tip to isolate a biological molecule. 
     In more particular aspects, the presently disclosed subject matter provides a high throughput method for identifying a protein, glycoprotein, or a glycan in a plurality of samples, the method comprising: (a) providing a plurality of samples comprising at least one protein comprising at least one peptide amino group or at least one glycoprotein comprising at least one oxidized glycan or at least one reactive groups of amino acid side chains or protein modifications; (b) disposing the plurality of samples in a plurality of pipette tips, wherein each pipette tip comprises an elongate body having a proximal end adapted to connect to and be in fluid communication with an outlet of a fluid dispensing device and a distal end having an opening adapted to dispense a fluid, the elongate body further comprising a fluid path between the proximal end and the distal end, wherein the fluid path comprises: (i) a first frit proximate the distal end and a second frit proximate the proximal end, and wherein the fluid path comprises a solid phase disposed between the first frit and the second frit, the solid phase comprising a chemical moiety capable of conjugating one or more glycoproteins through one or more oxidized glycans or an amino-reactive moiety capable of conjugating one or more amino groups or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications of one or more proteins disposed in the fluid path between the first frit and the second frit; or (ii) a monolith-bonded aldehyde-reactive chemical moiety or a monolith-bonded amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications; (c) conjugating the at least one protein or at least one glycoprotein comprising the plurality of samples to the solid phase chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications or the monolith-bonded aldehyde-reactive chemical moiety or amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications; (d) cleaving the at least one protein thereby releasing at least one peptide fragment or releasing the at least one former glycopeptide fragment or glycan; and (e) analyzing the at least one peptide, glycan or the at least one former glycopeptide fragment to identify the protein, glycan from which the at least one peptide and glycan fragment was derived or to identify the glycoprotein from which the former glycopeptide fragment was derived; and wherein at least one step of the method is automated. 
     Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein: 
         FIGS. 1A-1B  show (A) an embodiment of the workflow of the presently disclosed formerly N-linked glycopeptide isolation using a hydrazide tip; and (B) a representative embodiment of the presently disclosed pipette tip comprising a aldehyde-reactive hydrazide moiety; 
         FIGS. 2A-2D  show an experiment for the determination of time required for coupling, trypsin digestion and PNGase F release on a tip: (A) coupling time course: oxidized bovine fetuin was pipetted through a hydrazide tip. Concentration of protein uncoupled was measured at various time points; (B) digestion time course: fetuin conjugated to a hydrazide tip was subjected to trypsin digestion. Concentration of non-glycopeptide released from glycoprotein conjugated on hydrazide tip was measured at various time points; (C) fetuin glycopeptides conjugated to hydrazide tip through N-linked glycans were released by PNGase F. Peptide released was measured at various time points; and (D) a representative MALDI spectra of formerly N-linked glycopeptides from fetuin; (S): Signal to Noise ratio of each peak; 
         FIGS. 3A-3B  show Venn diagrams comparing the serum N-linked glycopeptide identified from three LC-MS/MS replicates and three isolation replicates. The diagram illustrates similarities and differences in the peptides identified in (A) each of the three isolation replicates and (B) each of the three LC-MS/MS replicates (Injection 1=333, Injection 2=341, Injection 3=332) by Proteome Discoverer software searches (Thermo Fisher Scientific, Waltham, Mass.) of MS/MS data; 
         FIGS. 4A-4B  show liquid chromatography profiles of serum N-linked glycopeptide from three LC-MS/MS replicates and three isolation replicates. The raw files of (A) the three LC-MS/MS replicates or (B) the three isolation replicates were displayed in Xcalibur and the base peak profiles were overlaid for visualization of LC variability; 
         FIGS. 5A-5C  show an embodiment of the scheme for Chemical Immobilization of Proteins for Peptide Extraction (CIPPE). Proteins are conjugated onto the solid support. Unbound compounds including OCT are washed away. Peptides are released from the solid support using proteolysis and analyzed using LC-MS/MS: (A, B) an embodiment of the workflow of the presently disclosed immobilization of proteins on a solid phase in a tip and releasing of peptides for global proteomics analysis from proteins immobilized in an amino-reactive resin in a tip; and (C) an embodiment of the workflow of the presently disclosed conjugation of proteins on a solid phase in a tip and releasing glycans from glycoproteins for glycomic analysis; 
         FIGS. 6A-6C  show mass spectrometric detection of the tryptic peptides from HSA with and without OCT: A) a representative electrospray ionization (ESI) spectrum of the tryptic peptides from OCT contaminated HSA digested in solution; B) a representative mass spectrum of OCT contaminated HSA after OCT removal using CIPPE; and C) a representative mass spectrum of clean HSA digested in solution; 
         FIG. 7  shows a schematic diagram for the relative quantification to study the impact of OCT on tissue samples using CIPPE method. Mouse kidney was split into two pieces. One was embedded in OCT, and second was directly frozen at −80° C. Proteins were extracted from two OCT-embedded tissues and one frozen tissue using CIPPE. Peptides were labeled with iTRAQ tags and labeled peptides were combined. Peptide sample was then divided into two fractions and 90% of sample was used for glycopeptide extraction using the SPEG method. The iTRAQ labeled tryptic peptides and glycopeptides were analyzed using LC-MSMS; 
         FIGS. 8A-8B  show quantitative analysis of proteins and glycoproteins isolated from OCT-embedded tissues using CIPPE. Scatter plot represents proteome (A) and glycoproteome (B). The two channels 114 and 115 were quantitative analysis of two OCT embedded tissues using CIPPE. The intensities observed for peptides in channels 114 and 115 were plotted in X axis and Y axis respectively for each PSM. Scatter plot represents quantitative linearity between reporter ion groups, the sample and the reporter ion intensity scatter plot are grouped around a 45° line indicating symmetric distribution of fold change across the scatter plot; 
         FIGS. 9A-9D  show quantitative analysis of proteins and glycoproteins form OCT-embedded tissue and frozen tissue: (A) scatter plot representing proteome; (B) scatter plot representing the glycoproteome. Channel 114 represents OCT embedded tissue and 116 represents frozen tissue. The intensities observed for peptides in channels 114 and 116 are plotted in X axis and Y axis respectively. Scatter plot represents quantitative linearity between reporter ion groups, the sample and the reporter ion intensity scatter plot are grouped around a 45° line. The data shows symmetric distribution of fold changes across the scatter plot; (C) global proteomics plotted protein ratio log 2 (116/114) in Y axis and log 2 (115/114) in X axis; and (D) glycoprotein plotted similarly. The results are centered on origin indicating high quantitative similarity between OCT embedded tissue and frozen tissue analysis using CIPPE; 
         FIGS. 10A-10B  show representative MALDI spectra of released tryptic global peptides released from casein immobilized to solid phase by reductive amination with a mass range of 500-4000 using an embodiment of the tube digestion method and the tip method. K.EDVPSER (SEQ ID NO:355); K.AVPYPQR (SEQ ID NO:356) is a peptide from beta casein; 
         FIGS. 11A-11B  show representative MALDI spectra of released tryptic peptides from casein immobilized to solid phase in tip with a mass range of 900-1700 using an embodiment of the tube digestion method and the tip method. R.FFVAPFPEVFGK (SEQ ID NO:357) and R.YLGYLEQLLR (SEQ ID NO:358) are peptides from alpha-S1-casein; 
         FIGS. 12A-12B  show an embodiment of a workflow scheme of N-glycan isolation: (A) scheme of GIG isolation; and (B) scheme of GIG isolation using aldehyde tips. Proteins from samples were first immobilized onto beads/tip columns. Sialic acid was then modified with p-toluidine. The beads/tips were subsequently washed extensively in 1% formic acid, 1M NaCl, 10% acetonitrile, and water. N-glycans were finally released with PNGase F; 
         FIGS. 13A-13B  show an embodiment of aldehyde tips: (A) a photograph of a unpacked and packed aldehyde tip; and (B) a photograph of 96-well aldehyde tips loaded in a robotic liquid handling system for automated glycan extraction; 
         FIGS. 14A-14B  show optimization of reaction time for coupling and PNGase F release: (A) serum proteins were slowly pipetted through aldehyde tips for various amount of time. Complete coupling was achieved after 30 min reaction; and (B) after extensive washing and sialic acid labeling, the N-glycans from serum proteins were released from the aldehyde tips with PNGase F for various times. N-glycan was still releasing after 2 hours; 
         FIG. 15  shows MALDI-MS profiles of serum N-glycans isolated with aldehyde tips; 
         FIG. 16  shows representative MALDI profiles of three isolations of N-glycan from human serum. N-glycans from three human serum samples (20 μL each) were isolated in parallel using the aldehyde tips with a robotic liquid handling system; 
         FIG. 17  shows representative reproducibility of N-glycan isolation. Glycans shown in  FIG. 16  were quantified; 
         FIG. 18  shows an embodiment of the workflow of using p-toluidine to modify the acid component of proteins and sialylated glycans and quantifying of glycans and glycopeptides using MALDI-MS; 
         FIGS. 19A-19C  show N-glycans identified and quantified from SW1990 Cells using the method shown in  FIG. 18 : (A) heavy and light labeled cell mix; (B) light labeled cell mix, no ManNAc treatment; and (C) heavy labeled ManNAc treated cell; 
         FIG. 20  shows an embodiment of the workflow for glycopeptide analysis using basic reverse phase fractionation; 
         FIG. 21  shows an embodiment of the workflow of the presently disclosed conjugation of proteins on a solid phase in a tip. Sample preparation including labeling was automated using liquid handling robotic systems; 
         FIG. 22  shows results from the method shown in  FIG. 20 ; and 
         FIG. 23  shows quantitation of AFNSTLPTHAQHEK (SEQ ID NO: 354) CD44 glycopeptide with triattenary sialylated peptide. 
     
    
    
     DETAILED DESCRIPTION 
     The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. 
     The glycoproteome contains valuable information, such as biomarkers that may be discovered for disease diagnosis and monitoring. With the ever increasing performances of mass spectrometers, the emphasis is shifting to the sample preparation step for better throughput and reproducibility. In addition, a greater than ever number of samples are being processed and subjected to mass spectrometry analysis, calling for automation for high throughput sample preparation. Automation can minimize variability due to human errors, provide greater consistency and reduce sample preparation time and effort. Therefore, to meet the pressing need in the mass spectrometry field, the presently disclosed subject matter provides a novel pipette tip, such as a hydrazide tip, and methods for an integrated workflow of glycopolypeptide or polypeptide isolation using the tips. In some embodiments, with the presently disclosed tips and methods thereof, the processing time is decreased to less than 8 hours. In other embodiments, glycoprotein or protein isolation can be automated using a liquid handling robot system. 
     I. Pipette Tips 
     A. Pipette Tips 
       FIG. 1A  shows, in some embodiments, the workflow of the presently disclosed formerly N-linked glycopeptide isolation using a hydrazide tip. 
     Referring now to  FIG. 1B , in some embodiments, the presently disclosed subject matter provides a pipette tip  100 , which includes elongate body  110  having proximal end  120  adapted to connect to and be in fluid communication with an outlet of a fluid dispensing device (not shown) and distal end  130  having opening  140  adapted to dispense a fluid, the elongate body  110  further comprising fluid path  150  between proximal end  120  and distal end  130 , wherein fluid path  150  comprises first frit  170  proximate distal end  130  and second frit  160  proximate distal end  120 , and wherein fluid path  150  comprises solid phase  180  disposed between first frit  170  and second frit  160 , wherein the solid phase  180  comprises: (i) a chemical moiety capable of conjugating one or more glycoproteins through one or more oxidized glycans; or (ii) an amino-reactive moiety capable of conjugating one or more amino groups of one or more proteins disposed in the fluid path  150  between the first frit  170  and the second frit  160 ; or (iii) other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications disposed in the fluid path  150  between the first frit  170  and the second frit  160 . 
     The pipette tip can be any kind, shape, or size, depending on the amount of chemical or amino-reactive moiety required, the kind of automated apparatus used, and the like for the particular presently disclosed methods. A person with ordinary skill in the art will appreciate that standard sizes of pipette tips are commercially available, such as from 50 μL to 1000 μL. In a preferred embodiment, the pipette tips used are meant for automated pipetting functions so that the hydrazide pipette tips can be used for high throughput methods. 
     In some embodiments, the presently disclosed subject matter provides a pipette tip comprising an elongate body having a proximal end adapted to connect to and be in fluid communication with an outlet of a fluid dispensing device and a distal end having an opening adapted to dispense a fluid, the elongate body further comprising a fluid path between the proximal end and the distal end, wherein the fluid path comprises: (a) a first frit proximate the distal end and a second frit proximate the proximal end, and wherein the fluid path comprises a solid phase disposed between the first frit and the second frit, the solid phase comprising: (i) a chemical moiety capable of conjugating one or more glycoproteins through one or more oxidized glycans; or (ii) an amino-reactive moiety capable of conjugating one or more amino groups of one or more proteins disposed in the fluid path between the first frit and the second frit; or (iii) other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications disposed in the fluid path between the first frit and the second frit; or (b) a monolith-bonded aldehyde-reactive chemical moiety, a monolith-bonded amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications. 
     The solid phase comprising a chemical moiety, such as a hydrazide moiety or an amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications, can be, for example, a bead, resin, slurry, monolith, membrane or disk, or any generally solid phase material suitable for the presently disclosed methods. An advantage of using a solid phase is that it allows extensive washing to remove undesired molecules. Another advantage of the solid phase is that it allows further manipulation of the sample molecules without the need for additional purification steps that can result in loss of sample molecules. In some embodiments, the chemical moiety is selected from the group consisting of one or more aldehyde-reactive hydrazide beads/resin/monolith or amino-reactive beads/resin/monolith or beads/resin/monolith with other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications. In other embodiments, the aldehyde-reactive chemical moiety is used for glycan conjugation and the amino-reactive moiety is used for polypeptide conjugation or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications. 
     Frits, also known as filters, are available in a wide variety of porous plastics such as polyethylene (PE), polytetrafluoroethylene (PTFE), oleophobic-treated PTFE, functionalized and surface-modified porous materials, bio-activated porous media, and the like. As used herein, in some embodiments, the frits hold the solid phase comprising an aldehyde-reactive chemical moiety or amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications in place and help protect the medium from running dry under buffer flow. 
     In some embodiments, the pipette tip comprises hydrazide resin. In other embodiments, the hydrazide resin has a particle size ranging from about 40 micrometers to about 60 micrometers. In further embodiments, the particle size range of the hydrazide resin is about 75 micrometers to about 300 micrometers. In still further embodiments, the first frit and the second frit have a pore size ranging from about 15 to about 45 microns. 
     In some embodiments, the pipette tip comprises more than two frits, such as 3, 4, 5, or more frits. 
     B. Methods for Preparing Pipette Tips 
     Referring again to  FIG. 1B , in some embodiments, the presently disclosed subject matter provides methods for preparing a pipette tip  100 . In some embodiments, the method comprises pushing a first frit  170  into elongate body  110 , adding a solid phase  180  to the elongate body  110  from the proximal end  120 , pushing a second frit  160  through the proximal end  120  to secure the solid phase  180  between the two frits  160  and  170 , wherein adding a solid phase  180  to the elongate body  110  comprises forming a fluid path  150  between the proximal end  120  and the distal end  130 . Forming a fluid path  150  comprises one of: (i) disposing a first frit  170  proximate the distal end  130  of the pipette tip  100  and disposing thereon a solid phase  180  comprising one of a chemical moiety capable of conjugating one or more glycoproteins through one or more oxidized glycans or an amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications capable of conjugating one or more amino groups of one or more proteins, and disposing a second frit  160  proximate the proximal end  120  of the pipette tip  100 ; or (ii) disposing a monolith-bonded aldehyde-reactive chemical moiety or a monolith-bonded amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications between the distal end  130  and the proximal end  120  of the pipette tip  100 . 
     In some embodiments, the presently disclosed subject matter provides a method for preparing a pipette tip, the method comprising: (a) providing a pipette tip comprising an elongate body having a proximal end adapted to connect to and be in fluid communication with an outlet of a fluid dispensing device and a distal end having an opening adapted to dispense a fluid; and (b) forming a fluid path between the proximal end and the distal end by one of: (i) disposing a first frit proximate the distal end of the pipette tip and disposing thereon a solid phase comprising one of a chemical moiety capable of conjugating one or more glycoproteins through one or more oxidized glycans or an amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications capable of conjugating one or more amino groups of one or more proteins, and disposing a second frit proximate the proximal end of the pipette tip; or (ii) disposing a monolith-bonded aldehyde-reactive chemical moiety or a monolith-bonded amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications between the distal end and the proximal end of the pipette tip. In some embodiments, the chemical moiety comprises an aldehyde-reactive moiety. In other embodiments, the first frit and the second frit have a pore size ranging from about 15 to about 45 microns. In still other embodiments, the methods further comprise washing the solid phase after the solid phase is disposed on the first frit. In further embodiments, the methods further comprise washing the solid phase with a liquid selected from the group consisting of water and a buffer. 
     Pushing the frits into the pipette tip can be performed with any tool that will allow the frit to be placed into the pipette tip, such as a tweezer, a needle, and the like. Likewise, there are different ways that the solid phase can be added to the pipette tip, such as using a pipetter and another pipette tip, a dropper, a micro capillary pipette, and the like. 
     C. Kits Comprising Pipette Tips 
     In general, a presently disclosed kit contains some or all of the components, reagents, supplies, and the like to practice a method according to the presently disclosed subject matter. In some embodiments, the presently disclosed subject matter provides a kit comprising at least one presently disclosed pipette tip, wherein the kit further comprises a set of instructions for using the at least one pipette tip to isolate a biological molecule. 
     II. Methods for Identifying Proteins and Glycoproteins 
     Protein glycosylation has long been recognized as a very common post-translational modification. Carbohydrates are linked to serine or threonine residues (O-linked glycosylation) or to asparagine residues (N-linked glycosylation). Protein glycosylation, and in particular N-linked glycosylation, is prevalent in proteins destined for extracellular environments. These include proteins on the extracellular side of the plasma membrane, secreted proteins, and proteins contained in body fluids, for example, blood serum, cerebrospinal fluid, urine, breast milk, saliva, lung lavage fluid, pancreatic juice, and the like. In some embodiments, the plurality of samples is selected from the group consisting of a body fluid, a secreted protein, and a cell surface protein. 
     The presently disclosed subject matter provides methods for quantitative profiling of glycoproteins and glycopeptides on a proteome-wide scale. The methods allow the identification and quantification of glycoproteins in a complex sample and determination of the sites of glycosylation. The methods can be used to determine changes in the abundance of glycoproteins and changes in the state of glycosylation at individual glycosylation sites on those glycoproteins that occur in response to perturbations of biological systems and organisms in health and disease. 
     The presently disclosed methods can be used to purify glycosylated proteins or peptides and identify and quantify the glycosylation sites. In some embodiments, because the methods can be directed to isolating glycoproteins, the methods also reduce the complexity of analysis since many proteins and fragments of glycoproteins do not contain carbohydrate. This can simplify the analysis of complex biological samples such as serum. The methods are advantageous for the determination of protein glycosylation in glycome studies and can be used to isolate and identify glycoproteins from cell membrane or body fluids to determine specific glycoprotein changes related to certain disease states or cancer. The methods can be used for detecting quantitative changes in protein samples containing glycoproteins and to detect their extent of glycosylation. The methods can be used for identifying oligosaccharides in samples. The methods are applicable for the identification and/or characterization of diagnostic biomarkers, immunotherapy, or other diagnostic or therapeutic applications. The methods can also be used to evaluate the effectiveness of drugs during drug development, optimal dosing, toxicology, drug targeting, and related therapeutic applications. 
     The presently disclosed tips and methods can be used to identify many different types of glycoproteins, glycans or proteins. These include mucins, collagens, antibodies, molecules of the major histocompatibility complex (MHC), viral glycoproteins, hormones, transport molecules, such as transferrin and ceruloplasmin, enzymes, various proteins involved in cell interactions with other cells, a virus, a bacterium, or a hormone, plasma proteins, calnexin, calreticulin, fetuin, casein, proteins involved in the regulation of development, specific glycoproteins on the surface membranes of platelets, and the like. 
     In some embodiments, the presently disclosed subject matter provides a high throughput method for identifying a protein, glycoprotein, or a glycan in a plurality of samples, the method comprising: (a) providing a plurality of samples comprising at least one protein comprising at least one peptide amino group or at least one glycoprotein comprising at least one oxidized glycan or at least one reactive groups of amino acid side chains or protein modifications; (b) disposing the plurality of samples in a plurality of pipette tips, wherein each pipette tip comprises an elongate body having a proximal end adapted to connect to and be in fluid communication with an outlet of a fluid dispensing device and a distal end having an opening adapted to dispense a fluid, the elongate body further comprising a fluid path between the proximal end and the distal end, wherein the fluid path comprises: (i) a first frit proximate the distal end and a second frit proximate the proximal end, and wherein the fluid path comprises a solid phase disposed between the first frit and the second frit, the solid phase comprising a chemical moiety capable of conjugating one or more glycoproteins through one or more oxidized glycans or an amino-reactive moiety capable of conjugating one or more amino groups or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications of one or more proteins disposed in the fluid path between the first frit and the second frit; or (ii) a monolith-bonded aldehyde-reactive chemical moiety or a monolith-bonded amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications; (c) conjugating the at least one protein or at least one glycoprotein comprising the plurality of samples to the solid phase chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications or the monolith-bonded aldehyde-reactive chemical moiety or amino-reactive moiety or other chemical moieties capable of conjugating to one or more reactive groups of amino acid side chains or protein modifications; (d) cleaving the at least one protein thereby releasing at least one peptide fragment or releasing the at least one former glycopeptide fragment or glycan; and (e) analyzing the at least one peptide, glycan or the at least one former glycopeptide fragment to identify the protein, glycan from which the at least one peptide and glycan fragment was derived or to identify the glycoprotein from which the former glycopeptide fragment was derived; and wherein at least one step of the method is automated. In other embodiments, the chemical moiety comprises a hydrazide moiety. In still other embodiments, the hydrazide moiety comprises a hydrazide resin. 
     The use of biological fluids, such as a body fluid as a sample source, is particularly useful for the presently disclosed methods. Biological fluid specimens are generally readily accessible and available in relatively large quantities for clinical analysis. Biological fluids can be used to analyze diagnostic and prognostic markers for various diseases. In addition to ready accessibility, body fluid specimens do not require any prior knowledge of the specific organ or the specific site in an organ that might be affected by disease. Because body fluids, in particular blood, are in contact with numerous body organs, body fluids “pick up” molecular signatures indicating pathology due to secretion or cell lysis associated with a pathological condition. Body fluids also pick up molecular signatures that are suitable for evaluating drug dosage, drug targets and/or toxic effects, as disclosed herein. In some embodiments, the plurality of samples is selected from the group consisting of samples comprising a body fluid, a secreted protein, and a cell surface protein. 
     The carbohydrate moieties of a glycoprotein are chemically or enzymatically modified to generate a reactive group that can be selectively bound to a solid support or solid phase having a corresponding reactive group. In some embodiments, at least one glycoprotein is oxidized with periodate. For example, the cis-diol groups of carbohydrates in glycoproteins can be oxidized by periodate oxidation to give a di-aldehyde, which reacts with a hydrazide moiety to form covalent hydrazone bonds. The hydroxyl groups of a carbohydrate can also be derivatized by epoxides or oxiranes, alkyl halogen, carbonyldiimidazoles, N,N′-disuccinimidyl carbonates, N-hydroxycuccinimidyl chloroformates, and the like. The hydroxyl groups of a carbohydrate can also be oxidized by enzymes to create reactive groups such as aldehyde groups. For example, galactose oxidase oxidizes terminal galactose or N-acetyl-D-galactose residues to form C-6 aldehyde groups. These derivatized groups can be conjugated to hydrazide-containing moieties. 
     In some embodiments, after being oxidized, at least one glycoprotein or protein is removed from the oxidation buffer and disposed in a coupling buffer. In other embodiments, the coupling buffer is a high salt and acidic pH buffer. In still other embodiments, the presently disclosed methods further comprise adding aniline to the coupling buffer. Aniline can be used as a catalyst to improve the reaction rate between aldehyde and hydrazide groups (Zeng et al., 2009; Dirksen et al., 2010). 
     After the samples are oxidized, they are added to the pipette tips for immobilization of the glycoproteins and/or the proteins. In some embodiments, the methods further comprise washing the at least one protein or the at least one glycoprotein with a urea buffer before being reduced. 
     If desired, the bound glycoproteins or proteins can be denatured and optionally reduced. Denaturing and/or reducing the bound glycoproteins or proteins can be useful prior to cleavage of the glycoproteins or proteins, in particular protease cleavage, because this allows access to protease cleavage sites that can be masked in the native form of the glycoproteins or proteins. The bound glycoproteins or proteins can be denatured with detergents and/or chaotropic agents. Reducing agents such as β-mercaptoethanol, dithiothreitol, tris-carboxyethylphosphine (TCEP), and the like, can also be used, if desired. The binding of the glycoproteins or proteins to a solid phase allows the denaturation step to be carried out followed by extensive washing to remove denaturants that could inhibit the enzymatic or chemical cleavage reactions. The use of denaturants and/or reducing agents can also be used to dissociate protein complexes in which non-glycosylated proteins form complexes with bound glycoproteins. Thus, the use of these agents can be used to increase the specificity for glycoproteins by washing away non-glycosylated proteins from the solid phase. In some embodiments, the at least one protein or the at least one glycoprotein is reduced with tris(2-carboxyethyl) phosphine (TCEP). 
     In some embodiments, at least one protein or glycoprotein is alkylated. In other embodiments, the at least one protein or the at least one glycoprotein is alkylated with iodoacetamide (IAA). In still other embodiments, the methods further comprise washing the at least one alkylated protein or the at least one alkylated glycoprotein with a urea buffer before being cleaved. 
     The bound glycoproteins or proteins can be cleaved into peptide fragments to facilitate analysis. Thus, a protein molecule can be enzymatically cleaved with one or more proteases into peptide fragments. Exemplary proteases useful for cleaving polypeptides include trypsin, chymotrypsin, pepsin, papain,  Staphylococcus aureus  (V8) protease,  Submaxillaris  protease, bromelain, thermolysin, and the like. In certain applications, proteases having cleavage specificities that cleave at fewer sites, such as sequence-specific proteases having specificity for a sequence rather than a single amino acid, can also be used, if desired. Polypeptides can also be cleaved chemically, for example, using CNBr, acid or other chemical reagents. One skilled in the art can readily determine appropriate conditions for cleavage to achieve a desired efficiency of peptide cleavage. In some embodiments, the at least one alkylated protein or the at least one alkylated glycoprotein is cleaved with trypsin. 
     However, in other embodiments, cleavage of the bound glycoproteins or proteins is not required, in particular where the bound glycoprotein is relatively small and contains a single glycosylation site. Furthermore, the cleavage reaction can be carried out after binding of glycoproteins to the solid phase, allowing characterization of non-glycosylated peptide fragments derived from the bound glycoprotein. Alternatively, the cleavage reaction can be carried out prior to addition of the glycoproteins to the solid phase. One skilled in the art can readily determine the desirability of cleaving the sample polypeptides and an appropriate point to perform the cleavage reaction, as needed for a particular application. 
     In some embodiments, cleaving the at least one alkylated glycoprotein comprising at least one oxidized glycan occurs by enzymatic reaction if the at least one oxidized glycan is an N-glycan or by chemical reaction if the at least one oxidized glycan is an O-glycan. In other embodiments, cleaving of the at least one alkylated protein occurs by using a protease or a chemical. In still other embodiments, cleaving of the at least one alkylated protein leaves at least one glycopeptide on the solid phase or monolith. In further embodiments, before releasing the at least one former glycopeptide fragment, the solid phase or monolith is washed to remove the non-glycosylated peptide fragments. 
     In some embodiments, the at least one former glycopeptide fragment is released from the solid phase or monolith with a glycosidase or chemicals. In other embodiments, the glycosidase is selected from the group consisting of an N-glycosidase and a β-elimination. In still other embodiments, the N-glycosidase is peptide-N-glycosidase F (PNGase F). In further embodiments, at least one former glycopeptide fragment is released from the solid phase using a chemical cleavage. 
     In some embodiments, the glycoproteins or proteins are isotopically labeled, for example, at the amino or carboxyl termini to allow the quantities of glycoproteins or proteins from different biological samples to be compared. 
     After isolating the glycoproteins, glycans or proteins from a sample and cleaving the glycoprotein or protein into fragments, the former glycopeptide, glycan or peptide fragments are released from the solid phase and the released former glycopeptide, glycan or peptide fragments are identified and/or quantified. A particularly useful method for analysis of the released glycopeptide or peptide fragments is mass spectrometry. A variety of mass spectrometry systems can be employed in the methods of the invention for identifying and/or quantifying a sample molecule such as a released glycopeptide or peptide fragment. Mass analyzers with high mass accuracy, high sensitivity and high resolution include, but are not limited to, ion trap, triple quadrupole, and time-of-flight, quadrupole time-of-flight mass spectrometers and Fourier transform ion cyclotron mass analyzers (FT-ICR-MS). Mass spectrometers are typically equipped with matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI) ion sources, although other methods of peptide ionization can also be used. In ion trap MS, analytes are ionized by ESI or MALDI and then put into an ion trap. Trapped ions can then be separately analyzed by MS upon selective release from the ion trap. Fragments can also be generated in the ion trap and analyzed. Sample molecules such as released glycopeptide or peptide fragments can be analyzed, for example, by single stage mass spectrometry with a MALDI-TOF or ESI-TOF system. Methods of mass spectrometry analysis are well known to those skilled in the art. In some embodiments, analyzing of the at least one glycopeptide fragment or the at least one former peptide fragment is done by mass spectrometry. 
     Once a peptide is analyzed by mass spectrometry, for example, the resulting CID spectrum can be compared to databases for the determination of the identity of the isolated glycopeptide or peptide. In particular, it is possible that one or a few peptide fragments can be used to identify a parent polypeptide from which the fragments were derived if the peptides provide a unique signature for the parent polypeptide. Thus, identification of a single glycopeptide, alone or in combination with knowledge of the site of glycosylation, can be used to identify a parent glycopolypeptide from which the glycopeptide fragments were derived. Further information can be obtained by analyzing the nature of the attached tag and the presence of the consensus sequence motif for carbohydrate attachment. For example, if peptides are modified with an N-terminal tag, each released glycopeptide or peptide has the specific N-terminal tag, which can be recognized in the fragment ion series of the CID spectra. Furthermore, the presence of a known sequence motif that is found, for example, in N-linked carbohydrate-containing peptides, that is, the consensus sequence NXS/T, can be used as a constraint in database searching of N-glycosylated peptides. 
     In addition, the identity of the parent glycopolypeptide or polypeptide can be determined by analysis of various characteristics associated with the peptide, for example, its resolution on various chromatographic media or using various fractionation methods. These empirically determined characteristics can be compared to a database of characteristics that uniquely identify a parent polypeptide, which defines a peptide tag. 
     In some embodiments, the method is automated, which allows many samples to be analyzed at the same time. Automated systems for testing or analyzing many samples simultaneously are known in the art. In other embodiments, the method further comprises the use of a liquid handling robot system. 
     III. General Definitions 
     Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs. 
     As used herein, the term “polypeptide” or “protein” refers to a peptide or polypeptide of two or more amino acids. A polypeptide can also be modified by naturally occurring modifications such as post-translational modifications, including phosphorylation, fatty acylation, prenylation, sulfation, hydroxylation, acetylation, addition of carbohydrate, addition of prosthetic groups or cofactors, formation of disulfide bonds, proteolysis, assembly into macromolecular complexes, and the like. A “peptide fragment” is a peptide of two or more amino acids, generally derived from a larger polypeptide. 
     As used herein, a “glycopolypeptide”, “glycopeptide” or “glycoprotein” refers to a polypeptide that contains a covalently bound carbohydrate group in the intact glycoproteins and could be released free of glycans from the glycoproteins before mass spectrometric analysis. The carbohydrate can be a monosaccharide, oligosaccharide or polysaccharide. Proteoglycans are included within the meaning of “glycopolypeptide.” A glycopolypeptide can additionally contain other post-translational modifications. A “glycopeptide” refers to a peptide that comprises a covalently bound carbohydrate. A “glycopeptide fragment” refers to a peptide fragment resulting from enzymatic or chemical cleavage of a larger polypeptide in which the peptide fragment retains covalently bound carbohydrate. It is understood that a glycopeptide fragment or peptide fragment refers to the peptides that result from a particular cleavage reaction, regardless of whether the resulting peptide was present before or after the cleavage reaction. Thus, a peptide that does not contain a cleavage site will be present after the cleavage reaction and is considered to be a peptide fragment resulting from that particular cleavage reaction. For example, if bound glycopeptides are cleaved, the resulting cleavage products retaining bound carbohydrate are considered to be glycopeptide fragments. The glycosylated fragments can remain bound to the solid phase, and such bound glycopeptide fragments are considered to include those fragments that were not cleaved due to the absence of a cleavage site. 
     As disclosed herein, a glycopolypeptide or glycopeptide can be processed such that the carbohydrate is removed from the parent glycopolypeptide. It is understood that such an originally glycosylated polypeptide is still referred to herein as a glycopolypeptide or glycopeptide even if the carbohydrate is removed enzymatically and/or chemically. Thus, a glycopolypeptide or glycopeptide can refer to a glycosylated or de-glycosylated form of a polypeptide. A glycopolypeptide or glycopeptide from which the carbohydrate is removed is referred to as the de-glycosylated form of a polypeptide whereas a glycopolypeptide or glycopeptide which retains its carbohydrate is referred to as the glycosylated form of a polypeptide. 
     As used herein, the term “glycan” refers to a polysaccharide or oligosaccharide. Glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan. As used herein, an “oxidized glycan” is a polysaccharide or an oligosaccharide that has been oxidized. 
     As used herein, a “hydrazide moiety” is a moiety comprising an acyl derivative of hydrazine. 
     As used herein, the term “amino-reactive moiety” is a moiety that can conjugate the amino groups of proteins. 
     As used herein, the term “aldehyde-reactive chemical moiety” is a moiety that can conjugate the aldehyde of a glycan. 
     As used herein, the term “monolith” is intended to mean a separation media that generally does not contain interparticular voids. As a result, the mobile phase flows through the stationary phase. 
     As used herein, the term “sample” is intended to mean any biological fluid, cell, tissue, organ or portion thereof, which includes one or more different molecules such as nucleic acids, polypeptides, or small molecules. A sample can be a tissue section obtained by biopsy, or cells that are placed in or adapted to tissue culture. A sample can also be a biological fluid specimen such as blood, serum or plasma, cerebrospinal fluid, urine, saliva, seminal plasma, pancreatic juice, breast milk, lung lavage, and the like. A sample can additionally be a cell extract from any species, including prokaryotic and eukaryotic cells as well as viruses. A tissue or biological fluid specimen can be further fractionated, if desired, to a fraction containing particular cell types. 
     As used herein, a “polypeptide sample” refers to a sample containing two or more different polypeptides. A polypeptide sample can include tens, hundreds, or even thousands or more different polypeptides. A polypeptide sample can also include non-protein molecules so long as the sample contains polypeptides. A polypeptide sample can be a whole cell or tissue extract or can be a biological fluid. Furthermore, a polypeptide sample can be fractionated using well known methods into partially or substantially purified protein fractions. 
     As used herein, the term “biological molecule” refers to any molecule found within a cell or produced by a living organism, including viruses. This term may include, but is not limited to, nucleic acids, polypeptides, carbohydrates, and lipids. A biological molecule can be isolated from various samples such as tissues of all kinds, cultured cells, body fluids, whole blood, blood serum, plasma, urine, feces, microorganisms, viruses, plants, and mixtures comprising nucleic acids following enzyme reactions. Examples of tissues include tissue from invertebrates, such as insects and mollusks, vertebrates such as fish, amphibians, reptiles, birds, and mammals such as humans, rats, dogs, cats and mice. Cultured cells can be from procaryotes, such as bacteria, blue green algae, actinomycetes, and mycoplasma and from eucaryotes, such as plants, animals, fungi, and protozoa. Blood samples include blood taken directly from an organism or blood that has been filtered in some way to remove some elements such as red blood cells, and/or serum or plasma. Nucleic acid can be isolated from enzyme reactions to purify the nucleic acid from enzymes such as DNA polymerase, RNA polymerase, reverse transcriptase, ligases, restriction enzymes, DNase, RNase, nucleases, proteases, and the like, or any other enzyme that can contact nucleic acids in a molecular biology method. Genomic DNA can be considered to be a “large biological molecule”. 
     Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth. 
     Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. 
     For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ±100% in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions. 
     Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range. 
     EXAMPLES 
     The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods. 
     Example 1 
     Rapid Analysis of N-Glycoproteome of Human Serum and Peptide Isolation by Conjugation to Amino-Linking Beads 
     Materials and Methods 
     Materials. 
     Hydrazide resin and sodium periodate were from Bio-Rad (Hercules, Calif.). BCA protein assay kit, Zeba spin desalting column (7k MWCO), Urea, and tris(2-carboxyethyl) phosphine (TCEP) were from Thermo Fisher Scientific (Waltham, Mass.). Sequencing-grade trypsin was from Promega (Madison, Wis.). PNGase F was from New England Biolabs (Ipswich, Mass.). alpha-CHC matrix was from Agilent Technology (Santa Clara, Calif.). Frits were from POREX (Fairburn, Ga.). All other chemicals were from Sigma-Aldrich (St. Louis, Mo.). 
     Preparation of Hydrazide Pipette Tip. 
     A round frit (2-mm-diameter and 1-mm-thick, pore size 15-45 microns) was first pushed into the pipette tip end (Disposable Automation Research Tips, Thermo Fisher Scientific, Waltham, Mass.). Two hundred microliters of hydrazide resin (50% slurry) was then loaded into each pipette tip. Liquids were blown out of the tip and a 5-mm round frit was pushed into the tip to secure the hydrazide resin between the two frits. The tips were then washed 5 times with 200 μL of water and conditioned 5 times with coupling buffer (100-mM sodium acetate, 1-M sodium chloride, pH 5.5) by aspirating and dispensing the solution. For less than 5% of the prepared tips, the flow was too slow due to high resistance, and the tips were therefore discarded. 
     Coupling Time for Glycoprotein to Hydrazide Tip. 
     Four hundred microliters of bovine fetuin in oxidation buffer (500 mM sodium acetate, 0.3 mM sodium chloride, pH 5) was oxidized with 15 mM sodium periodate for 1 h at room temperature in the dark followed by buffer exchange into coupling buffer. After addition of 100-mM aniline, the fetuin samples were slowly pipetted through hydrazide tips for coupling. Aliquots of fetuin samples were saved before, as well as after, fetuin was coupled for 1, 5, 10, 20, 30, 60 and 120 min. Protein concentration was determined using the BCA protein assay per manufacturer&#39;s protocol after removal of aniline. The absorbance was read at 562 nm with a spectrophotometer (BioTek, Winooski, Vt.). The results were plotted against time and data presented represent mean±SD (n=3). 
     Incubation Time for Trypsin Digestion. Bovine fetuin coupled to the hydrazide tips through oxidized glycans was washed with 3-mL urea buffer (8-M urea in 0.4-M NH 4 HCO 3 ), reduced with 10-mM TCEP for 30 min, and alkylated with 12-mM iodoacetamide (IAA) for 15 min in the dark at room temperature (RT). After washing again with 3-mL urea buffer, the conjugated fetuin was digested with trypsin (1:30) in 100-mM ammonium bicarbonate where the digested non-glycopeptides were released into trypsin solution. Aliquots of trypsin solutions were saved before and after the samples were digested for 1, 5, 10, 20, 30, 60 and 120 min. The peptide concentration in each aliquot was then determined by BCA protein assay. The results were plotted against time and data presented represent mean±SD (n=3). 
     Incubation Time for PNGase F Release. 
     After digestion, the hydrazide tips (with conjugated glycopeptides) were washed extensively with 6-mL solutions of 1.5-M sodium chloride, 80% acetonitrile (ACN), deionized (DI) water, and 25-mM ammonium bicarbonate buffer to remove any residual non-glycopeptides released by digestion. 1500 U of PNGase F in 200 μL of 25-mM ammonium bicarbonate was then pipetted through the hydrazide tips. Aliquots of PNGase F solutions (with released peptides) were saved before and after releasing of any residual non-glycopeptides for 1, 5, 10, 20, 30, 60 and 120 min. A 10 −12  M angiotensin I standard in 50% ACN/1% TFA was used to serve as an internal standard. An equal amount of angiotensin I standard and samples (three sets of fetuin glycopeptides collected at various times of PNGase F incubations) were applied to matrix-assisted laser desorption/ionization (MALDI) spots, coated with alpha-CHC matrix and analyzed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) (4800, AB SCIEX, Framingham, Mass.). A total of 20 subspectra (100 shots/subspectrum) were averaged to yield the mass spectrum for each sample. Area under the curve for angiotensin I and the major fetuin glycopeptide released (LCPDCPLLAPLNDSR; SEQ ID NO:1) were recorded. The ratio of fetuin/angiotensin was calculated and plotted against time. Data presented represent mean±SD (n=3). 
     Isolation of N-linked Glycopeptides from Human Serum with a Hydrazide Tip. 
     N-linked glycopeptides were isolated from human serum using a hydrazide tip similar to that described above. Briefly, 40 μL of human serum (n=3) was diluted 1:1 with oxidation buffer, oxidized with sodium periodate, and buffer exchanged into coupling buffer. The serum sample was then slowly aspirated into hydrazide tips and dispensed back into a 96-well plate for 30 min using a liquid handling robotic system (Versette, Thermo Fisher Scientific, Waltham, Mass.). The aspiration and dispensing were repeated during the entire incubation time. The glycoproteins captured in the hydrazide tips were then reduced, alkylated, and digested for 1 h by pipetting the tips through TCEP, IAA and trypsin solutions (1:120 based on initial protein amount). The tips were then washed extensively and glycopeptides were released with 1500 U PNGase F in 25-mM ammonium bicarbonate buffer for 1 h at RT. Tips were then washed three times with 50% ACN and the eluents were combined and vacuumed to dryness. Samples were resuspended with 40 μl 5% ACN/0.2% formic acid. Two microliters of each sample were injected into a Q-Exactive mass spectrometer (Q-E, Thermo Fisher Scientific, Waltham, Mass.) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. 
     LC-MS/MS Analysis. 
     Formerly N-linked glycopeptides were analyzed using a Q-E mass spectrometer with an EASY-Spray source. Peptides were separated with a 15-cm×75-μm C18 column on an Ultimate 3000 series UHPLC at a flow rate of 300 nL/min with a 110 min linear gradient (from 5 to 35% B over 75 min; A=0.1% formic acid 2% ACN in water, B=0.1% formic acid in 90% ACN). Full mass spectrometry (MS) scans were acquired over the mass range 400-1800 m/z with a mass resolution of 70,000. The AGC target value was set at 3,000,000. The fifteen most intense peaks were fragmented with Higher-energy Collisional Dissociation (HCD) with collision energy of 27. MS/MS was acquired with a resolution of 17,500 with an AGC target of 50,000 and max injection time of 200 ms. Dynamic exclusion was set for 15 sec. 
     Identification of Glycosites and Glycopeptide Quantification. 
     The resulting MS/MS spectra were searched against the European Bioinformatics Institute (http://www.ebi.ac.uk/) non-redundant International Protein Index human sequence database (IPI, v3.87, 2011/09/27, 91,491 entries) using Proteome Discoverer (v 1.4, Thermo Fisher Scientific, Waltham, Mass.). Base peak profiles of the three LC-MS/MS replicates or the three isolation replicates were opened and overlaid using the Xcalibur software (Thermo Fisher Scientific, Waltham, Mass.). For peptide identification, a mass tolerance of 10 ppm was permitted for intact peptide masses and 0.6 Da for HCD-fragmented ions, with allowance for two missed cleavages in the trypsin digests, oxidized methionine, and deamidated asparagine as potential variable modifications. Carboxyamidomethylation (C) was set as a fixed modification. Peptides with 1% FDR were reported with their peptide spectrum match (PSM). Peptides with N-glycosites (NXS/T, where X can be any amino acid except P) were required. For N-linked glycopeptides commonly identified in all three LC-MS/MS replicates or in all three isolation replicates, coefficient of variation (CV) for each peptide was calculated based on PSM; total PSMs was also calculated for each peptide by adding up the PSMs recorded in each run. The average CV for peptides with total PSMs over 150, between 150 and 60, between 60 and 30, between 30 and 15 and below 15 were calculated. 
     Workflow Using the Presently Disclosed Hydrazide Tip 
     To achieve high throughput N-linked glycopeptide enrichment from serum, the presently disclosed subject matter provides a hydrazide tip for fast and reproducible N-linked glycopeptide isolation through solid phase extraction.  FIG. 1  shows the flowchart of N-linked glycopeptide isolation with hydrazide tips above eppendorf tubes. Briefly, serum comprising proteins with oxidized glycans were pipetted through hydrazide tips in the presence of 100 mM aniline. Glycoproteins in the serum were conjugated covalently to the hydrazide resin packed in the tips. Glycoproteins captured on the tips were then denatured, reduced, alkylated, and digested by aspirating and dispensing the hydrazide tips in urea, TCEP, IAA and trypsin solution, respectively. The tips were then washed extensively with 1.5-M sodium chloride, 80% ACN, deionized (DI) water, and 25-mM ammonium bicarbonate buffer to removed residual non-glycopeptides. Finally, the formerly N-linked glycopeptides were released by pipetting the hydrazide tips in PNGase solution. 
     Incubation Times for the Major Steps of the Presently Disclosed Methods Using a Hydrazide Tip 
     To determine the reaction times of the major steps of the presently disclosed methods, i.e. coupling, proteolysis and PNGase F release for glycopeptide capture of serum, bovine fetuin, a 38 kD glycoprotein with three N-linked glycosylation sites, was used as a standard. 
     To determine the incubation time required for complete coupling of the glycoproteins to the hydrazide tip, 0.8 mg oxidized bovine fetuin proteins were coupled with hydrazide tips in the presence of 100-mM aniline for various times. The amount of fetuin used here equals the amount of glycoprotein estimated from 40 μL of human serum. Aniline was used as a catalyst to improve the reaction rate between aldehyde and hydrazide groups as previously reported (Zeng et al., 2009; Dirksen et al., 2010). It was found that essentially no fetuin was present in the solution at 10 min, suggesting that coupling was complete after a 10 min incubation ( FIG. 2A ). 
     To determine the incubation time required for trypsin digestion, the fetuin proteins coupled to the hydrazide tips above were denatured, reduced, and alkylated. The fetuin samples were then digested with trypsin using a trypsin-to-glycoprotein ratio of 1:30 for various times. This trypsin-to-glycoprotein ratio was also used in the serum glycopeptide isolation where glycoproteins account for about 25% of the total serum proteins. It was found that no additional peptides were released into the trypsin solutions after 1 h, suggesting that trypsin digestion was complete at 1 h ( FIG. 2B ). 
     To determine the incubation time required for PNGase F release of formerly N-linked glycopeptides, the hydrazide tips were washed extensively with 1.5-M sodium chloride, 80% ACN, DI water, and 25-mM ammonium bicarbonate buffer to remove any residual non-glycopeptides released by digestion. PNGase F in 25-mM ammonium bicarbonate was then pipetted through the hydrazide tips for various times. Again, the PNGase F-to-glycoprotein ratio is similar to that used in serum glycopeptide isolations. As shown in  FIG. 2C , most peptides were released after 1 h. At this time point, all three predicted formerly N-linked glycopeptides of fetuin (LCPDCPLLAPLNDSR, SEQ ID NO:1; VVHAVEVALATFNAESNGSYLQLVEISR, SEQ ID NO:2; and RPTGEVYDIEIDTLETTCHVLDPTPLANCSVR, SEQ ID NO:3) could be observed by MALDI-TOF-TOF ( FIG. 2D ). 
     Thus, with the presently disclosed hydrazide tip and methods thereof, the total time required to complete N-linked glycopeptide isolation was within 8 h. The hydrazide tip contains hydrazide resins 40-60 micrometers in size with 0.1-μm micropores. After packing, the spacing between resins is estimated to be roughly 50-90 micrometers considering a face-centered cubic or hexagonal close-packed arrangement (Conway et al., 1999). Without wishing to be bound to any one particular theory, it is believed that such small dimensions enable the presently disclosed hydrazide tips to work as a microfluidic reactor, where the reaction rate is significantly improved due to faster mixing (Sia and Whitesides, 2003). As shown above, the presently disclosed methods decreased the processing time to less than 8 hours. In addition, the isolation capacity could be easily adjusted by simply controlling the amount of hydrazide beads packed into each tip. As the loading capacity of the hydrazide beads is about 40-μL serum/200-μL hydrazide beads (50% slurry) as previously reported (Zhou et al., 2007), the hydrazide beads packed could be adjusted accordingly for optimal performance when a different amount of serum needs to be processed. Moreover, the presently disclosed workflow methods provided herein could be used to isolate N-linked glycopeptides in diverse types of samples, such as body fluids. Finally, as the presently disclosed hydrazide tip could be readily used in liquid handling robotic systems, in some embodiments, the presently disclosed methods provide automation of N-linked glycopeptide isolation for high throughput sample preparation. 
     Rapid Analysis of N-Glycoproteome of Human Serum 
     To attempt automation of isolation of N-linked glycopeptides, the hydrazide tips were used in combination with a liquid handling robotic system to perform glycopeptide isolation from human serum. Forty microliters of serum was processed with each hydrazide tip and 1/20th glycopeptide isolated was injected into a Q-E mass spectrometer for LC-MS/MS analysis. 
     Table 1 shows the identification, specificity and missed cleavage of glycopeptides isolated using hydrazide tip and the original SPEG procedure. Formerly N-linked glycopeptides were isolated from 40 μL of human serum with the presently disclosed methods using a hydrazide tip or with the original SPEG method. 1/20th of the glycopeptides isolated was injected into a QE mass spectrometer for LC-MS/MS analysis. The number and specificity of formerly N-linked glycopeptides identified as well as the percentage of peptides with missed cleavage were listed for each isolation. 
     After controlling the FDR&lt;1% for peptide identification, 332, 345 and 328 unique formerly N-linked glycopeptides from human serum were identified in Isolations 1, 2, and 3, respectively (Table 1). In comparison, a similar number of unique glycopeptides, 315, was identified from the same human serum when the isolation was carried out using the original SPEG isolation method (Zhang et al., 2003). The specificity of N-linked glycopeptides identified was also similar between the hydrazide tip isolations (89.04%, 86.59% and 90.07%) and the original SPEG isolation method (81.66%). The missed cleavages observed were 20.22%, 21.07% and 20.30%, for Isolations 1, 2, and 3, respectively, and 16.38% for the original SPEG isolation method. 
     Table 2 shows the unique formerly N-linked glycopeptides of human serum identified in three isolation replicates. Human serum samples were subjected to N-linked glycopeptide isolation with the presently disclosed hydrazide tips. An aliquot of the formerly N-linked glycopeptides from each isolation (n=3) was injected once into a Q-E mass spectrometer for LC-MS/MS analysis. The sequences of the unique peptides identified are listed with their peptide spectral match (PSM). 
     Table 3 shows the unique formerly N-linked glycopeptides of human serum identified in three LC-MS/MS Replicates. Human serum samples were subjected to N-linked glycopeptide isolation with the presently disclosed hydrazide tips. An aliquot of the formerly N-linked glycopeptides was injected three times into a Q-E mass spectrometer for LC-MS/MS analysis. The sequences of the unique peptides identified are listed with their peptide spectral match (PSM). 
     Altogether, a total of 379 unique formerly N-linked glycopeptides were identified in the three isolation replicates with 294 commonly identified ( FIG. 3A ; Table 2). Similarly, a total of 366 unique formerly N-linked glycopeptides were identified in the three LC-MS/MS replicates, with 306 of them commonly identified ( FIG. 3B ; Table 3). In both cases, the commonly identified peptides were about 80% of that totally identified. In addition, great consistency was observed in the LC profiles between the LC-MS/MS replicates and the isolation replicates ( FIG. 4 ). 
     Table 4 shows the reproducibility of glycopeptide isolations using the presently disclosed hydrazide tip. Formerly N-linked glycopeptides from 40 μl human serum were isolated three times in parallel with the presently disclosed methods and hydrazide tip. 1/20th of the glycopeptides isolated from Isolation 1 was injected three times into a QE mass spectrometer for LC-MS/MS analysis; 1/20th of the glycopeptides isolated from Isolations 2 and 3 was injected once into a QE mass spectrometer for LC-MS/MS analysis. The MS/MS spectra generated were searched against human IPI 3.87 for identification of glycopeptides. Peptide spectral matches (PSMs) reported for each glycopeptide were used to calculate the coefficient of variations (CVs) between injections and between isolations. The CVs were listed along with the total number of PSMs added up from each run. 
     Table 4 shows that the reproducibility between isolation replicates was comparable to that between LC-MS/MS replicates, with CVs, based on the PSMs, only slightly higher between isolations (Table 4). Overall, the CVs increased as the PSM of glycopeptides decreased as reported before (Liu et al., 2004). The CVs between isolations were 6.32%, 11.36%, 9.98%, 17.01% and 28.1% for glycopeptides with a total PSM over or equal to 150, between less than 150 and more than or equal to 60, between less than 60 and more than or equal to 30, between less than 30 and more than or equal to 15, and less than 15, respectively. In comparison, the CVs between LC-MS/MS replicates were 4.53%, 6.27%, 8.57%, 11.53% and 21.55% for glycopeptides with a total PSM over or equal to 150, between less than 150 and more than or equal to 60, between less than 60 and more than or equal to 30, between less than 30 and more than or equal to 15, and less than 15. These data demonstrate that glycopeptide isolation with hydrazide tips has high throughput, great reproducibility, and automation capability when used in combination with liquid handling robotic systems. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Identification, Specificity and Missed Cleavage of Glycopeptides 
               
               
                 Isolated Using Hydrazide Tip and the Original SPEG Procedure 
               
            
           
           
               
               
               
               
            
               
                   
                 Glycopeptides 
                   
                 Missed 
               
               
                 Sample  
                 Identified 
                 Specificity  
                 Cleavage 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Hydrazide Tip Isolation 1 
                 332 
                 89.04% 
                 20.22% 
               
               
                 Hydrazide Tip Isolation 2 
                 345 
                 86.59% 
                 21.07% 
               
               
                 Hydrazide Tip Isolation 3 
                 328 
                 90.07% 
                 20.30% 
               
               
                 Original SPEG Isolation  
                 315 
                 81.66% 
                 16.38% 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Unique Formerly N-linked Glycopeptides of Human Serum Identified in 
               
               
                 Three Isolation Replicates. 
               
            
           
           
               
               
               
            
               
                   
                 SEQ 
                 Peptide Spectrum Match 
               
            
           
           
               
               
               
               
               
            
               
                   
                 ID 
                 Isolation 
                 Isolation 
                 Isolation 
               
               
                 Sequence 
                 NO: 
                 1 
                 2 
                 3 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 AALAAFnAQNnGSnFQLEEISR 
                 4 
                 119 
                 121 
                 105 
               
               
                   
               
               
                 AATcINPLnGSVcERPAnHSAK 
                 5 
                 1 
                 3 
                 2 
               
               
                   
               
               
                 ADGTVnQIEGEATPVnLTEPAK 
                 6 
                 19 
                 21 
                 21 
               
               
                   
               
               
                 ADGTVNQIEGEATPVnLTEPAKLEVK 
                 7 
                 13 
                 14 
                 11 
               
               
                   
               
               
                 ADTHDEILEGLNFnLTEIPEAQIH 
                 8 
                 8 
                 6 
                 6 
               
               
                   
               
               
                 ADTHDEILEGLnFnLTEIPEAQIHEGFQELLR 
                 9 
                 117 
                 118 
                 119 
               
               
                   
               
               
                 AELSnHTRPVILVPGcLGNQLEAK 
                 10 
                 2 
                 3 
                 2 
               
               
                   
               
               
                 AFEnVTDLQWLILDHnLLEnSK 
                 11 
                 18 
                 15 
                 11 
               
               
                   
               
               
                 AFHYnVSSHGcQLLPWTQHSPHTR 
                 12 
                 2 
                 1 
                 #N/A 
               
               
                   
               
               
                 AFITnFSMIIDGmTYPGIIK 
                 13 
                 7 
                 5 
                 5 
               
               
                   
               
               
                 AFITnFSMIIDGmTYPGIIKEK 
                 14 
                 3 
                 3 
                 2 
               
               
                   
               
               
                 AGAFLGLTNVAVmnLSGNcLR 
                 15 
                 8 
                 9 
                 6 
               
               
                   
               
               
                 AGLQAFFQVQEcnK 
                 16 
                 7 
                 7 
                 6 
               
               
                   
               
               
                 AHLnVSGIPcSVLLADVEDLIQQQISnDTVSPR 
                 17 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 ALPQPQnVTSLLGcTH 
                 18 
                 5 
                 7 
                 16 
               
               
                   
               
               
                 ALQAVYSmmSWPDDVPPEGWnR 
                 19 
                 1 
                 3 
                 #N/A 
               
               
                   
               
               
                 AMMAFTADLFSLVAQTSTcPNLILSPLSVALALSHLALGAQnHTLQR 
                 20 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 AnLSSQALQmSLDYGFVTPLTSmSIR 
                 21 
                 13 
                 12 
                 11 
               
               
                   
               
               
                 APDKNVIFSPLSISTALAFLSLGAHnTTLTEILK 
                 22 
                 9 
                 9 
                 10 
               
               
                   
               
               
                 AQLLQGLGFnLTER 
                 23 
                 18 
                 17 
                 20 
               
               
                   
               
               
                 AQVIInITDVDEPPIFQQPFYHFQLK 
                 24 
                 2 
                 4 
                 3 
               
               
                   
               
               
                 AREDIFMETLKDIVEYYnDSnGSHVLQGR 
                 25 
                 21 
                 22 
                 24 
               
               
                   
               
               
                 AVLQLnEEGVDTAGSTGVTLnLTSKPIILR 
                 26 
                 37 
                 34 
                 34 
               
               
                   
               
               
                 AVnITSENLIDDVVSLIR 
                 27 
                 10 
                 11 
                 8 
               
               
                   
               
               
                 AYLLPAPPAPGnASESEEDR 
                 28 
                 3 
                 4 
                 3 
               
               
                   
               
               
                 cATPHGDnASLEATFVK 
                 29 
                 3 
                 4 
                 3 
               
               
                   
               
               
                 cGLVPVLAENYnK 
                 30 
                 4 
                 6 
                 7 
               
               
                   
               
               
                 cGncSLTTLKDEDFcK 
                 31 
                 7 
                 7 
                 6 
               
               
                   
               
               
                 cGncSLTTLKDEDFcKR 
                 32 
                 7 
                 8 
                 7 
               
               
                   
               
               
                 cIQAnYSLmENGK 
                 33 
                 21 
                 21 
                 21 
               
               
                   
               
               
                 cIQAnYSLmEnGKIK 
                 34 
                 2 
                 1 
                 3 
               
               
                   
               
               
                 cmWSSALnSLnLSFAGLEQVPK 
                 35 
                 3 
                 4 
                 1 
               
               
                   
               
               
                 cSDGWSFDATTLDDnGTmLFFK 
                 36 
                 19 
                 26 
                 16 
               
               
                   
               
               
                 DFVnASSKYEITTIHnLFR 
                 37 
                 3 
                 6 
                 4 
               
               
                   
               
               
                 DHEnGTGTNTYAALNSVYLmmNNQmR 
                 38 
                 6 
                 10 
                 7 
               
               
                   
               
               
                 DIVEYYNDSnGSHVLQGR 
                 39 
                 37 
                 36 
                 30 
               
               
                   
               
               
                 DKIcDLLVANNHFAHFFAPQnLTNmNK 
                 40 
                 31 
                 31 
                 35 
               
               
                   
               
               
                 DmTEVISSLENAnYKDHENGTGTNTYAALNSVYLMMNNQMR 
                 41 
                 6 
                 8 
                 8 
               
               
                   
               
               
                 DQcIVDDITYNVnDTFHK 
                 42 
                 11 
                 11 
                 11 
               
               
                   
               
               
                 DQcIVDDITYNVnDTFHKR 
                 43 
                 2 
                 1 
                 1 
               
               
                   
               
               
                 DRQDGEEVLQcmPVcGRPVTPIAQnQTTLGSSR 
                 44 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 DSVSVVLGQHFFnR 
                 45 
                 3 
                 3 
                 5 
               
               
                   
               
               
                 DTAVFEcLPQHAmFGNDTITcTTHGnWTK 
                 46 
                 32 
                 34 
                 25 
               
               
                   
               
               
                 DTAVFEcLPQHAmFGnDTITcTTHGnWTKLPEcR 
                 47 
                 10 
                 7 
                 10 
               
               
                   
               
               
                 DVQIIVFPEDGIHGFnFTR 
                 48 
                 7 
                 8 
                 8 
               
               
                   
               
               
                 EDIFmETLKDIVEYYnDSNGSHVLQGR 
                 49 
                 4 
                 4 
                 4 
               
               
                   
               
               
                 EEQYNSTYRVVSVLTVLHQDWLnGK 
                 50 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 EEQYnSTYRVVSVLTVLHQDWLnGKEYK 
                 51 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 EGDHEFLEVPEAQEDVEATFPVHQPGnYScSYR 
                 52 
                 17 
                 17 
                 19 
               
               
                   
               
               
                 EGYSnISYIVVNHQGISSR 
                 53 
                 9 
                 10 
                 10 
               
               
                   
               
               
                 EHEAQSnASLDVFLGHTNVEELmK 
                 54 
                 9 
                 10 
                 5 
               
               
                   
               
               
                 EHEGAIYPDnTTDFQR 
                 55 
                 26 
                 24 
                 27 
               
               
                   
               
               
                 EHETcLAPELYNGnYSTTQK 
                 56 
                 5 
                 5 
                 5 
               
               
                   
               
               
                 EHYnLSAATcSPGQmcGHYTQVVWAK 
                 57 
                 2 
                 2 
                 2 
               
               
                   
               
               
                 ELDREVYPWYnLTVEAK 
                 58 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 ELHHLQEQnVSNAFLDK 
                 59 
                 27 
                 25 
                 27 
               
               
                   
               
               
                 ELHHLQEQnVSnAFLDKGEFYIGSK 
                 60 
                 94 
                 74 
                 79 
               
               
                   
               
               
                 ELPGVcnETmmALWEEcKPcLK 
                 61 
                 10 
                 14 
                 8 
               
               
                   
               
               
                 EnLTAPGSDSAVFFEQGTTR 
                 62 
                 16 
                 15 
                 16 
               
               
                   
               
               
                 ERSWPAVGncSSALR 
                 63 
                 2 
                 2 
                 1 
               
               
                   
               
               
                 EVnTSGFAPARPPPQPGSTTFWAWSVLR 
                 64 
                 5 
                 5 
                 4 
               
               
                   
               
               
                 EVSFLncSLDnGGcTHYcLEEVGWR 
                 65 
                 5 
                 6 
                 7 
               
               
                   
               
               
                 EVYPWYnLTVEAK 
                 66 
                 2 
                 3 
                 1 
               
               
                   
               
               
                 EWEKELHHLQEQnVSnAFLDKGEFYIGSK 
                 67 
                 5 
                 2 
                 3 
               
               
                   
               
               
                 EYESYSDFERnVTEK 
                 68 
                 1 
                 1 
                 2 
               
               
                   
               
               
                 FcRDnYTDLVAIQNK 
                 69 
                 2 
                 #N/A 
                 2 
               
               
                   
               
               
                 FDFQGTcEYLLSAPcHGPPLGAEnFTVTVAnEHR 
                 70 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 FEDGVLDPDYPRnISDGFDGIPDNVDAALALPAHSYSGR 
                 71 
                 7 
                 10 
                 9 
               
               
                   
               
               
                 FEVDSPVYnATWSASLK 
                 72 
                 3 
                 4 
                 4 
               
               
                   
               
               
                 FGHSAVLHnSTMYVFGGFNSLLLSDILVFTSEQcDAHR 
                 73 
                 5 
                 8 
                 6 
               
               
                   
               
               
                 FHDVSESTHWTPFLnASVHYIR 
                 74 
                 6 
                 6 
                 6 
               
               
                   
               
               
                 FLNnGTcTAEGK 
                 75 
                 3 
                 1 
                 3 
               
               
                   
               
               
                 FLTEVEKnATALYHVEAFK 
                 76 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 FnFQGTcEYLLSAPcHGPPLGAEnFTVTVAnEHR 
                 77 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 FnLTETSEAEIHQSFQHLLR 
                 78 
                 182 
                 173 
                 175 
               
               
                   
               
               
                 FNPGAESVVLSnSTLK 
                 79 
                 1 
                 1 
                 2 
               
               
                   
               
               
                 FnSSYLQGTNQITGR 
                 80 
                 2 
                 1 
                 1 
               
               
                   
               
               
                 FQSPAGTEALFELHNISVADSAnYScVYVDLKPPFGGSAPSER 
                 81 
                 93 
                 79 
                 82 
               
               
                   
               
               
                 FSDGLESnSSTQFEVK 
                 82 
                 52 
                 63 
                 59 
               
               
                   
               
               
                 FSDGLESnSSTQFEVKK 
                 83 
                 3 
                 3 
                 3 
               
               
                   
               
               
                 FSLLGHASIScTVEnETIGVWRPSPPTcEK 
                 84 
                 73 
                 71 
                 65 
               
               
                   
               
               
                 FSYSKnETYQLFLSYSSK 
                 85 
                 10 
                 10 
                 10 
               
               
                   
               
               
                 FVGTPEVnQTTLYQR 
                 86 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 FVQAIcEGDDcQPPAYTYNnITcASPPEVVGLDLR 
                 87 
                 6 
                 3 
                 2 
               
               
                   
               
               
                 FVQGnSTEVAcHPGYGLPK 
                 88 
                 4 
                 3 
                 3 
               
               
                   
               
               
                 GAFISnFSMTVDGK 
                 89 
                 9 
                 9 
                 16 
               
               
                   
               
               
                 GcnDSDVLAVAGFALR 
                 90 
                 3 
                 3 
                 4 
               
               
                   
               
               
                 GcScFSDWQGPGcSVPVPAnQSFWTR 
                 91 
                 4 
                 6 
                 2 
               
               
                   
               
               
                 GcVLLSYLnETVTVSASLESVR 
                 92 
                 132 
                 129 
                 129 
               
               
                   
               
               
                 GDSGGPLVcmDANnVTYVWGVVSWGEncGKPEFPGVYTK 
                 93 
                 12 
                 12 
                 13 
               
               
                   
               
               
                 GETHEQVHSILHFKDFVnASSK 
                 94 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 GETHEQVHSILHFKDFVnASSKYEITTIHNLFR 
                 95 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 GFGVAIVGnYTAALPTEAALR 
                 96 
                 49 
                 54 
                 48 
               
               
                   
               
               
                 GFLALYQTVAVnYSQPISEASR 
                 97 
                 7 
                 7 
                 6 
               
               
                   
               
               
                 GGETAQSADPQWEQLNNKnLSmPLLPADFHK 
                 98 
                 13 
                 15 
                 11 
               
               
                   
               
               
                 GGnSnGALcHFPFLYNNHnYTDcTSEGR 
                 99 
                 14 
                 14 
                 15 
               
               
                   
               
               
                 GGNSNGALcHFPFLYNnHnYTDcTSEGRR 
                 100 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 GHFIYKnVSEDLPLPTFSPTLLGDSR 
                 101 
                 1 
                 3 
                 #N/A 
               
               
                   
               
               
                 GLKFnLTETSEAEIHQSFQHLLR 
                 102 
                 86 
                 90 
                 71 
               
               
                   
               
               
                 GLnLTEDTYKPR 
                 103 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 GLTFQQnASSmcGPDQDTAIR 
                 104 
                 8 
                 10 
                 11 
               
               
                   
               
               
                 GLTFQQnASSmcVPDQDTAIR 
                 105 
                 9 
                 10 
                 11 
               
               
                   
               
               
                 GmnLTVFGGTVTAFLGIPYAQPPLGR 
                 106 
                 4 
                 1 
                 2 
               
               
                   
               
               
                 GNEANYYSnATTDEHGLVQFSInTTnVmGTSLTVR 
                 107 
                 134 
                 124 
                 128 
               
               
                   
               
               
                 GNVAVTVSGHTcQHWSAQTPHTHnR 
                 108 
                 9 
                 11 
                 8 
               
               
                   
               
               
                 GPSTPLPEDPnWnVTEFHTTPK 
                 109 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 GTAnTTTAGVPcQR 
                 110 
                 2 
                 2 
                 #N/A 
               
               
                   
               
               
                 GTGnDTVLNVALLNVISNQEcNIK 
                 111 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 GVTSVSQIFHSPDLAIRDTFVnASR 
                 112 
                 3 
                 4 
                 5 
               
               
                   
               
               
                 HAnWTLTPLK 
                 113 
                 5 
                 7 
                 6 
               
               
                   
               
               
                 HEEGHmLncTcFGQGR 
                 114 
                 6 
                 4 
                 6 
               
               
                   
               
               
                 HGIQYFnnNTQHSSLFmLnEVK 
                 115 
                 29 
                 41 
                 27 
               
               
                   
               
               
                 HGIQYFNnNTQHSSLFmLnEVKR 
                 116 
                 14 
                 13 
                 15 
               
               
                   
               
               
                 HGIQYFnnNTQHSSLFTLnEVK 
                 117 
                 40 
                 41 
                 40 
               
               
                   
               
               
                 HGIQYFnnNTQHSSLFTLNEVKR 
                 118 
                 11 
                 10 
                 10 
               
               
                   
               
               
                 HGVIISSTVDTYEnGSSVEYR 
                 119 
                 10 
                 11 
                 10 
               
               
                   
               
               
                 HLQmDIHIFEPQGISFLETESTFmTNQLVDALTTWQnK 
                 120 
                 5 
                 9 
                 6 
               
               
                   
               
               
                 HYLVSnISHDTVLQcHFTcSGK 
                 121 
                 3 
                 2 
                 3 
               
               
                   
               
               
                 HYTnSSQDVTVPcR 
                 122 
                 13 
                 13 
                 13 
               
               
                   
               
               
                 HYYIAAEEIIWnYAPSGIDIFTKEnLTAPGSDSAVFFEQGTTR 
                 123 
                 11 
                 10 
                 10 
               
               
                   
               
               
                 IADAHLDRVEnTTVYYLVLDVQESDcSVLSR 
                 124 
                 68 
                 58 
                 64 
               
               
                   
               
               
                 IcDLLVAnNHFAHFFAPQnLTNmnK 
                 125 
                 23 
                 21 
                 23 
               
               
                   
               
               
                 IDSTGnVTNELR 
                 126 
                 3 
                 5 
                 3 
               
               
                   
               
               
                 IITILEEEmnVSVcGLYTYGKPVPGHVTVSIcR 
                 127 
                 74 
                 67 
                 64 
               
               
                   
               
               
                 INNDFNYEFYnSTWSYVK 
                 128 
                 6 
                 6 
                 6 
               
               
                   
               
               
                 IPcSQPPQIEHGTInSSR 
                 129 
                 21 
                 19 
                 19 
               
               
                   
               
               
                 ISEEnETTcYMGK 
                 130 
                 14 
                 16 
                 14 
               
               
                   
               
               
                 ISnSSDTVEcEcSENWK 
                 131 
                 5 
                 4 
                 3 
               
               
                   
               
               
                 ISNSSDTVEcEcSEnWKGEAcDIPHcTDNcGFPHR 
                 132 
                 3 
                 5 
                 6 
               
               
                   
               
               
                 ITPnLAEFAFSLYRQLAHQSnSTnIFFSPVSIATAFAmLSLGTK 
                 133 
                 2 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 ITYSIVQTncSK 
                 134 
                 12 
                 12 
                 14 
               
               
                   
               
               
                 ITYSIVQTncSKEnFLFLTPDcK 
                 135 
                 11 
                 13 
                 11 
               
               
                   
               
               
                 IVGGTnSSWGEWPWQVSLQVK 
                 136 
                 8 
                 9 
                 6 
               
               
                   
               
               
                 IVLDPSGSMnlYLVLDGSDSIGASnFTGAK 
                 137 
                 134 
                 130 
                 132 
               
               
                   
               
               
                 IYPGVDFGGEELnVTFVK 
                 138 
                 7 
                 5 
                 7 
               
               
                   
               
               
                 IYSGILnLSDITK 
                 139 
                 7 
                 7 
                 8 
               
               
                   
               
               
                 IYSnHSALESLALIPLQAPLK 
                 140 
                 3 
                 4 
                 4 
               
               
                   
               
               
                 KAFITnFSMIIDGmTYPGIIK 
                 141 
                 4 
                 5 
                 4 
               
               
                   
               
               
                 KAFITnFSMIIDGmTYPGIIKEK 
                 142 
                 4 
                 7 
                 3 
               
               
                   
               
               
                 KcGncSLTTLKDEDFcK 
                 143 
                 1 
                 1 
                 2 
               
               
                   
               
               
                 KDFEDLYTPVnGSIVIVR 
                 144 
                 2 
                 2 
                 #N/A 
               
               
                   
               
               
                 KEHETcLAPELYNGnYSTTQK 
                 145 
                 12 
                 11 
                 12 
               
               
                   
               
               
                 KIVLDPSGSmnIYLVLDGSDSIGASnFTGAK 
                 146 
                 42 
                 35 
                 37 
               
               
                   
               
               
                 KLHINHNnLTESVGPLPK 
                 147 
                 6 
                 7 
                 6 
               
               
                   
               
               
                 KLINDYVKnGTR 
                 148 
                 2 
                 2 
                 2 
               
               
                   
               
               
                 KLPPGLLAnFTLLR 
                 149 
                 5 
                 6 
                 6 
               
               
                   
               
               
                 KnQSVNVFLGHTAIDEmLK 
                 150 
                 3 
                 5 
                 4 
               
               
                   
               
               
                 KQVHFFVnASDVDNVK 
                 151 
                 5 
                 5 
                 4 
               
               
                   
               
               
                 KVcQDcPLLAPLnDTR 
                 152 
                 12 
                 15 
                 16 
               
               
                   
               
               
                 LAGKPTHVnVSVVMAEVDGTcY 
                 153 
                 64 
                 59 
                 66 
               
               
                   
               
               
                 LAnLTQGEDQYYLR 
                 154 
                 15 
                 15 
                 15 
               
               
                   
               
               
                 LATALSLSNKFVEGSHnSTVSLTTK 
                 155 
                 2 
                 4 
                 2 
               
               
                   
               
               
                 LDAPTNLQFVnETDSTVLVR 
                 156 
                 9 
                 6 
                 6 
               
               
                   
               
               
                 LDPVSLQTLQTWnTSYPK 
                 157 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 LDREnISEYHLTAVIVDK 
                 158 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 LDREnISEYHLTAVIVDKDTGENLETPSSFTIK 
                 159 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 LEDLEVTGSSFLnLSTnIFSnLTSLGK 
                 160 
                 12 
                 18 
                 12 
               
               
                   
               
               
                 LEPVHLQLQcMSQEQLAQVAAnATK 
                 161 
                 12 
                 12 
                 12 
               
               
                   
               
               
                 LETTVnYTDSQRPIcLPSK 
                 162 
                 3 
                 4 
                 3 
               
               
                   
               
               
                 LFGDKSLTFnETYQDISELVYGAK 
                 163 
                 4 
                 5 
                 6 
               
               
                   
               
               
                 LGAcnDTLQQLMEVFK 
                 164 
                 34 
                 34 
                 35 
               
               
                   
               
               
                 LGAcnDTLQQLmEVFKFDTISEK 
                 165 
                 9 
                 10 
                 10 
               
               
                   
               
               
                 LGAcnDTLQQLMEVFKFDTISEKTSDQIHFFFAK 
                 166 
                 3 
                 4 
                 3 
               
               
                   
               
               
                 LGHcPDPVLVnGEFSSSGPVnVSDK 
                 167 
                 9 
                 10 
                 8 
               
               
                   
               
               
                 LGSFEGLVnLTFIHLQHNR 
                 168 
                 13 
                 11 
                 12 
               
               
                   
               
               
                 LGSLQELFLDSNnISELPPQVFSQLFcLER 
                 169 
                 3 
                 4 
                 4 
               
               
                   
               
               
                 LGSYPVGGnVSFEcEDGFILR 
                 170 
                 5 
                 7 
                 6 
               
               
                   
               
               
                 LGTSLSSGHVLMnGTLK 
                 171 
                 8 
                 10 
                 9 
               
               
                   
               
               
                 LHINHNnLTESVGPLPK 
                 172 
                 11 
                 10 
                 10 
               
               
                   
               
               
                 LKELPGVcnETMmALWEEcKPcLK 
                 173 
                 11 
                 20 
                 12 
               
               
                   
               
               
                 LLLSQLDSHPSHSAVVnWTSYASSIEALSSGNK 
                 174 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 LNAEnnATFYFK 
                 175 
                 108 
                 117 
                 103 
               
               
                   
               
               
                 LnDTLDYEcHDGYESnTGSTTGSIVcGYnGWSDLPIcYER 
                 176 
                 14 
                 13 
                 18 
               
               
                   
               
               
                 LNVEAAnWTVR 
                 177 
                 4 
                 3 
                 3 
               
               
                   
               
               
                 LPASLAEYTVTQLRPnATYSVcVmPLGPGR 
                 178 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 LPPGLLAnFTLLR 
                 179 
                 3 
                 6 
                 4 
               
               
                   
               
               
                 LPTQnITFQTESSVAEQEAEFQSPK 
                 180 
                 30 
                 34 
                 33 
               
               
                   
               
               
                 LPYQGnATmLVVLmEK 
                 181 
                 2 
                 1 
                 1 
               
               
                   
               
               
                 LQAILGVPWKDKncTSR 
                 182 
                 13 
                 13 
                 11 
               
               
                   
               
               
                 LQAPLnYTEFQKPIcLPSK 
                 183 
                 7 
                 8 
                 7 
               
               
                   
               
               
                 LQNnENnIScVER 
                 184 
                 7 
                 6 
                 8 
               
               
                   
               
               
                 LSDLSInSTEcLHVHcR 
                 185 
                 84 
                 72 
                 78 
               
               
                   
               
               
                 LSHnELADSGIPGNSFnVSSLVELDLSYNK 
                 186 
                 26 
                 20 
                 28 
               
               
                   
               
               
                 LSLHRPALEDLLLGSEAnLTcTLTGLR 
                 187 
                 92 
                 77 
                 85 
               
               
                   
               
               
                 LSSWVLLmKYLGnATAIFFLPDEGK 
                 188 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 LSVDKDQYVEPEnVTIQcDSGYGVVGPQSITcSGnR 
                 189 
                 5 
                 8 
                 3 
               
               
                   
               
               
                 LTDTIcGVGnmSAnASDQER 
                 190 
                 5 
                 7 
                 4 
               
               
                   
               
               
                 LVSANRLFGDKSLTFnETYQDISELVYGAK 
                 191 
                 3 
                 2 
                 4 
               
               
                   
               
               
                 LYHFLLGAWSLnATELDPcPLSPELLGLTK 
                 192 
                 15 
                 13 
                 8 
               
               
                   
               
               
                 LYLGSnnLTALHPALFQnLSK 
                 193 
                 12 
                 12 
                 11 
               
               
                   
               
               
                 mAGKPTHInVSVVmAEADGTcY 
                 194 
                 2 
                 3 
                 4 
               
               
                   
               
               
                 mAGKPTHVnVSVVmAEVDGTcY 
                 195 
                 3 
                 2 
                 4 
               
               
                   
               
               
                 mAWPEDHVFISTPSFnYTGR 
                 196 
                 6 
                 5 
                 4 
               
               
                   
               
               
                 MDGASnVTcINSR 
                 197 
                 25 
                 27 
                 20 
               
               
                   
               
               
                 MLLTFHTDFSNEEnGTImFYK 
                 198 
                 1 
                 3 
                 #N/A 
               
               
                   
               
               
                 mLNnNTGIYTcSAQGVWmNK 
                 199 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 mLnTSSLLEQLnEQFNWVSR 
                 200 
                 26 
                 35 
                 23 
               
               
                   
               
               
                 mPSQAPTGNFYPQPLLnSSmcLEDSR 
                 201 
                 4 
                 7 
                 4 
               
               
                   
               
               
                 mQcLAAALKDETnMSGGGEQADILPAnYVVKDR 
                 202 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 mSnITFLnFDPPIEEFHQYYQHIVTTLVK 
                 203 
                 1 
                 2 
                 #N/A 
               
               
                   
               
               
                 mVSHHnLTTGATLInEQWLLTTAK 
                 204 
                 443 
                 390 
                 483 
               
               
                   
               
               
                 mVSHHnLTTGATLINEQWLLTTAKNLFLnHSEnATAK 
                 205 
                 10 
                 11 
                 11 
               
               
                   
               
               
                 mVTAFTTccTLSEEFAcVDNLADLVFGELcGVNEnR 
                 206 
                 2 
                 4 
                 3 
               
               
                   
               
               
                 NAHGEEKEnLTAR 
                 207 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 ncGVncSGDVFTALIGEIASPnYPKPYPEnSR 
                 208 
                 8 
                 9 
                 9 
               
               
                   
               
               
                 NcQDIDEcVTGIHncSInETcFNIQGGFR 
                 209 
                 1 
                 2 
                 2 
               
               
                   
               
               
                 NEEYnKSVQEIQATFFYFTPnKTEDTIFLR 
                 210 
                 12 
                 11 
                 10 
               
               
                   
               
               
                 nEMLEIQVFNYSKVFSnK 
                 211 
                 2 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 nGTGHGnSTHHGPEYmR 
                 212 
                 2 
                 5 
                 6 
               
               
                   
               
               
                 NHPnITFFVYVSnFTWPIK 
                 213 
                 4 
                 4 
                 3 
               
               
                   
               
               
                 nISDGFDGIPDNVDAALALPAHSYSGR 
                 214 
                 2 
                 2 
                 1 
               
               
                   
               
               
                 nLASRPYTFHSHGITYYKEHEGAIYPDnTTDFQR 
                 215 
                 5 
                 4 
                 5 
               
               
                   
               
               
                 NLFLnHSEnATAK 
                 216 
                 243 
                 258 
                 279 
               
               
                   
               
               
                 NLFLnHSEnATAKDIAPTLTLYVGK 
                 217 
                 11 
                 11 
                 10 
               
               
                   
               
               
                 NLFLnHSEnATAKDIAPTLTLYVGKK 
                 218 
                 2 
                 3 
                 3 
               
               
                   
               
               
                 nnATVHEQVGGPSLTSDLQAQSK 
                 219 
                 51 
                 46 
                 31 
               
               
                   
               
               
                 NnmSFVVLVPTHFEWnVSQVLAnLSWDTLHPPLVWERPTK 
                 220 
                 2 
                 3 
                 1 
               
               
                   
               
               
                 NPPmGGNVVIFDTVITNQEEPYQnHSGR 
                 221 
                 5 
                 6 
                 9 
               
               
                   
               
               
                 NPVGLIGAEnATGETDPSHSK 
                 222 
                 11 
                 11 
                 11 
               
               
                   
               
               
                 nQALnLSLAYSFVTPLTSmVVTKPDDQEQSQVAEKPmEGESR 
                 223 
                 9 
                 12 
                 8 
               
               
                   
               
               
                 NSVLnSSTAEHSSPYSEDPIEDPLQPDVTGIR 
                 224 
                 3 
                 4 
                 4 
               
               
                   
               
               
                 nVIFSPLSISTALAFLSLGAHnTTLTEILK 
                 225 
                 11 
                 14 
                 11 
               
               
                   
               
               
                 QDQcIYnTTYLNVQR 
                 226 
                 181 
                 176 
                 169 
               
               
                   
               
               
                 QDQcIYnTTYLNVQREnGTISR 
                 227 
                 5 
                 5 
                 5 
               
               
                   
               
               
                 QEDLSVGSVLLTVnATDPDSLQHQTIR 
                 228 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 QGGVnATQVLIQHLR 
                 229 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 QInSSISGNLWDKDQR 
                 230 
                 3 
                 3 
                 2 
               
               
                   
               
               
                 QLAHQSnSTnIFFSPVSIATAFAmLSLGTK 
                 231 
                 92 
                 95 
                 80 
               
               
                   
               
               
                 QLDMLDLSnNSLASVPEGLWASLGQPNWDMR 
                 232 
                 16 
                 14 
                 9 
               
               
                   
               
               
                 QLEEFLnQSSPFYFWmNGDR 
                 233 
                 27 
                 28 
                 28 
               
               
                   
               
               
                 QLEEFLnQSSPFYFWmnGDRIDSLLEnDR 
                 234 
                 11 
                 13 
                 12 
               
               
                   
               
               
                 QLVEIEKVVLHPnYSQVDIGLIK 
                 235 
                 5 
                 3 
                 5 
               
               
                   
               
               
                 QNESHnFSGDIALLELQHSIPLGPNVLPVcLPDnETLYR 
                 236 
                 5 
                 6 
                 6 
               
               
                   
               
               
                 QnQcFYnSSYLnVQR 
                 237 
                 18 
                 16 
                 13 
               
               
                   
               
               
                 QPQAGLSQAnFTLGPVSR 
                 238 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 QQQHLFGSnVTDcSGnFcLFR 
                 239 
                 160 
                 159 
                 160 
               
               
                   
               
               
                 QVHFFVnASDVDNVK 
                 240 
                 12 
                 12 
                 11 
               
               
                   
               
               
                 QVLFLDTVYGncSTHFTVK 
                 241 
                 4 
                 6 
                 4 
               
               
                   
               
               
                 QVQVLQnLTTTYEIVLWQPVTADLIVK 
                 242 
                 2 
                 2 
                 3 
               
               
                   
               
               
                 REGDHEFLEVPEAQEDVEATFPVHQPGnYScSYR 
                 243 
                 12 
                 12 
                 14 
               
               
                   
               
               
                 RHEEGHmLncTcFGQGR 
                 244 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 RNPPmGGNVVIFDTVITNQEEPYQnHSGR 
                 245 
                 12 
                 15 
                 14 
               
               
                   
               
               
                 SDHGSSIScQPPAEIPGYLPADTVHLAVEFFnLTHLPANLLQGASK 
                 246 
                 10 
                 11 
                 9 
               
               
                   
               
               
                 SHAASDAPEnLTLLAETADAR 
                 247 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 SHEIWTHScPQSPGnGTDASH 
                 248 
                 2 
                 3 
                 1 
               
               
                   
               
               
                 SIPAcVPWSPYLFQPnDTcIVSGWGR 
                 249 
                 13 
                 13 
                 13 
               
               
                   
               
               
                 SKPTVSSSmEFKYDFnSSmLYSTAK 
                 250 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 SKWnITmESYVVHTnYDEYAIFLTK 
                 251 
                 19 
                 19 
                 15 
               
               
                   
               
               
                 SLGnVnFTVSAEALESQELcGTEVPSVPEHGR 
                 252 
                 157 
                 136 
                 146 
               
               
                   
               
               
                 SLGnVnFTVSAEALESQELcGTEVPSVPEHGRK 
                 253 
                 3 
                 1 
                 1 
               
               
                   
               
               
                 SLTFnETYQDISELVYGAK 
                 254 
                 29 
                 28 
                 28 
               
               
                   
               
               
                 SPYEMFGDEEVmcLNGnWTEPPQcK 
                 255 
                 31 
                 31 
                 27 
               
               
                   
               
               
                 SPYYnVSDEISFHcYDGYTLR 
                 256 
                 157 
                 151 
                 147 
               
               
                   
               
               
                 SQILEGLGFnLTELSESDVHR 
                 257 
                 20 
                 18 
                 20 
               
               
                   
               
               
                 SRVYLQGLIDcYLFGnSSTVLEDSK 
                 258 
                 1 
                 2 
                 2 
               
               
                   
               
               
                 SRYPHKPEInSTTHPGADLQENFcR 
                 259 
                 16 
                 16 
                 17 
               
               
                   
               
               
                 SSVITLnTnAELFnQSDIVAHLLSSSSSVIDALQYK 
                 260 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 STGKPTLYnVSLVMSDTAGTcY 
                 261 
                 6 
                 7 
                 7 
               
               
                   
               
               
                 SVQEIQATFFYFTPnKTEDTIFLR 
                 262 
                 104 
                 113 
                 101 
               
               
                   
               
               
                 SVVAPATDGGLnLTSTFLR 
                 263 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 TEGRPDmKTELFSSScPGGImLnETGQGYQR 
                 264 
                 2 
                 2 
                 4 
               
               
                   
               
               
                 TELFSSScPGGImLnETGQGYQR 
                 265 
                 12 
                 12 
                 12 
               
               
                   
               
               
                 TEVSSnHVLIYLDKVSnQTLSLFFTVLQDVPVR 
                 266 
                 7 
                 10 
                 8 
               
               
                   
               
               
                 TEVSSnHVLIYLDKVSnQTLSLFFTVLQDVPVRDLKPAIVK 
                 267 
                 2 
                 2 
                 2 
               
               
                   
               
               
                 THTnISESHPnATFSAVGEASIcEDDWnSGER 
                 268 
                 20 
                 25 
                 17 
               
               
                   
               
               
                 TKPREEQYnSTYR 
                 269 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 TLFcnASKEWDnTTTEcR 
                 270 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 TLnQSSDELQLSMGnAmFVK 
                 271 
                 206 
                 217 
                 210 
               
               
                   
               
               
                 TLYETEVFSTDFSnISAAK 
                 272 
                 9 
                 7 
                 9 
               
               
                   
               
               
                 TTTVQVPmMHQmEQYYHLVDmELncTVLQMDYSK 
                 273 
                 10 
                 9 
                 9 
               
               
                   
               
               
                 TVIRPFYLTnSSGVD 
                 274 
                 3 
                 4 
                 2 
               
               
                   
               
               
                 TVLTPATNHmGnVTFTIPANR 
                 275 
                 26 
                 19 
                 27 
               
               
                   
               
               
                 TVLTPATNHmGnVTFTIPAnREFK 
                 276 
                 2 
                 1 
                 1 
               
               
                   
               
               
                 TVVTYHIPQnSSLENVDSR 
                 277 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 TYNVLDmKnTTcQDLQIEVTVK 
                 278 
                 7 
                 5 
                 3 
               
               
                   
               
               
                 VASVININPnTTHSTGScR 
                 279 
                 3 
                 3 
                 2 
               
               
                   
               
               
                 VcQDcPLLAPLnDTR 
                 280 
                 25 
                 29 
                 30 
               
               
                   
               
               
                 VcQDcPLLAPLnDTRVVHAAK 
                 281 
                 6 
                 8 
                 9 
               
               
                   
               
               
                 VDKDLQSLEDILHQVEnK 
                 282 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 VEGSSSHLVTFTVLPLEIGLHNInFSLETWFGK 
                 283 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 VEnTTVYYLVLDVQESDcSVLSR 
                 284 
                 22 
                 15 
                 24 
               
               
                   
               
               
                 VFHIHnESWVLLTPK 
                 285 
                 4 
                 3 
                 3 
               
               
                   
               
               
                 VFPLSLDSTPQDGNVVVAcLVQGFFPQEPLSVTWSESGQnVTAR 
                 286 
                 12 
                 12 
                 9 
               
               
                   
               
               
                 VGQLQLSHnLSLVILVPQNLK 
                 287 
                 19 
                 17 
                 17 
               
               
                   
               
               
                 VIDFncTTSSVSSALANTK 
                 288 
                 18 
                 16 
                 18 
               
               
                   
               
               
                 VIDFncTTSSVSSALAnTKDSPVLIDFFEDTER 
                 289 
                 1 
                 1 
                 2 
               
               
                   
               
               
                 VLSnNSDANLELInTWVAK 
                 290 
                 54 
                 39 
                 33 
               
               
                   
               
               
                 VLTLNLDQVDFQHAGnYScVASNVQGK 
                 291 
                 2 
                 1 
                 1 
               
               
                   
               
               
                 VLYLAAYncTLRPVSK 
                 292 
                 7 
                 9 
                 8 
               
               
                   
               
               
                 VPGnVTAVLGETLK 
                 293 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 VPMmLQSSTISYLHDSELPcQLVQmNYVGnGTVFFILPDK 
                 294 
                 7 
                 12 
                 7 
               
               
                   
               
               
                 VSAITLVSATSTTAnmTVGPEGK 
                 295 
                 4 
                 3 
                 3 
               
               
                   
               
               
                 VSEHIPVYQQEEnQTDVWTLLNGSK 
                 296 
                 8 
                 9 
                 7 
               
               
                   
               
               
                 VSEHIPVYQQEEnQTDVWTLLnGSKDDFLIYDR 
                 297 
                 6 
                 8 
                 7 
               
               
                   
               
               
                 VSLTnVSISDEGR 
                 298 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 VSnQTLSLFFTVLQDVPVR 
                 299 
                 28 
                 23 
                 25 
               
               
                   
               
               
                 VSnVScQASVSR 
                 300 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 VSTVYANnGSVLQGTSVASVYHGK 
                 301 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 VTAcHSSQPnATLYK 
                 302 
                 7 
                 7 
                 7 
               
               
                   
               
               
                 VTISGVYDLGDVLEEmGIADLFTNQAnFSR 
                 303 
                 12 
                 13 
                 14 
               
               
                   
               
               
                 VTQnLTLIEESLTSEFIHDIDR 
                 304 
                 9 
                 7 
                 8 
               
               
                   
               
               
                 VTQVYAEnGTVLQGSTVASVYK 
                 305 
                 14 
                 13 
                 15 
               
               
                   
               
               
                 VTQVYAEnGTVLQGSTVASVYKGK 
                 306 
                 3 
                 3 
                 2 
               
               
                   
               
               
                 VTWKPQGAPVEWEEETVTnHTLR 
                 307 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 VVLHPnYSQVDIGLIK 
                 308 
                 29 
                 24 
                 31 
               
               
                   
               
               
                 VVLHPnYSQVDIGLIKLK 
                 309 
                 1 
                 #N/A 
                 2 
               
               
                   
               
               
                 VYIHPFHLVIHnESTcEQLAK 
                 310 
                 14 
                 13 
                 15 
               
               
                   
               
               
                 VYKPSAGnnSLYR 
                 311 
                 9 
                 7 
                 9 
               
               
                   
               
               
                 VYLQGLIDcYLFGnSSTVLEDSK 
                 312 
                 5 
                 8 
                 6 
               
               
                   
               
               
                 VYSGILnQSEIKEDTSFFGVQEIIIHDQYK 
                 313 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 WDPEVncSmAQIQLcPPPPQIPnSHnMTTTLNYR 
                 314 
                 30 
                 36 
                 31 
               
               
                   
               
               
                 WFSAGLASnSSWLR 
                 315 
                 2 
                 3 
                 1 
               
               
                   
               
               
                 WFYIASAFRNEEYnK 
                 316 
                 3 
                 3 
                 3 
               
               
                   
               
               
                 WnITmESYVVHTNYDEYAIFLTK 
                 317 
                 12 
                 11 
                 9 
               
               
                   
               
               
                 WNVNAPPTFHSEMMYDnFTLVPVWGK 
                 318 
                 3 
                 6 
                 #N/A 
               
               
                   
               
               
                 WVLTAAHcLLYPPWDKnFTENDLLVR 
                 319 
                 14 
                 15 
                 14 
               
               
                   
               
               
                 YAEDKFnETTEK 
                 320 
                 7 
                 9 
                 8 
               
               
                   
               
               
                 YFYnGTSmAcETFQYGGcmGnGNNFVTEK 
                 321 
                 25 
                 16 
                 22 
               
               
                   
               
               
                 YGNPNETQnnSTSWPVFK 
                 322 
                 4 
                 5 
                 3 
               
               
                   
               
               
                 YKGLnLTEDTYKPR 
                 323 
                 2 
                 2 
                 2 
               
               
                   
               
               
                 YLGnATAIFFLPDEGK 
                 324 
                 115 
                 102 
                 103 
               
               
                   
               
               
                 YLGnATAIFFLPDEGKLQHLEnELTHDIITK 
                 325 
                 58 
                 52 
                 63 
               
               
                   
               
               
                 YLHTAVIVSGTMLVFGGNTHnDTSmSHGAK 
                 326 
                 2 
                 3 
                 3 
               
               
                   
               
               
                 YnSQnQSNNQFVLYR 
                 327 
                 14 
                 10 
                 11 
               
               
                   
               
               
                 YnWSFIHcPAcQcnGHSK 
                 328 
                 3 
                 3 
                 #N/A 
               
               
                   
               
               
                 YPHKPEInSTTHPGADLQENFcR 
                 329 
                 17 
                 16 
                 19 
               
               
                   
               
               
                 YPPTVSmVEGQGEKnVTFWGRPLPR 
                 330 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 YQFNTNVVFSnnGTLVDR 
                 331 
                 9 
                 5 
                 6 
               
               
                   
               
               
                 YTcEEPYYYmEnGGGGEYHcAGnGSWVnEVLGPELPK 
                 332 
                 12 
                 10 
                 8 
               
               
                   
               
               
                 YTGnASALFILPDQDK 
                 333 
                 16 
                 15 
                 17 
               
               
                   
               
               
                 YTGnASALFILPDQDKmEEVEAmLLPETLK 
                 334 
                 25 
                 28 
                 26 
               
               
                   
               
               
                 YTGnASALFILPDQDKmEEVEAmLLPETLKR 
                 335 
                 31 
                 33 
                 30 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Unique Formerly N-linked Glycopeptides of Human Serum Identified in 
               
               
                 Three LC-MS/MS Replicates. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Peptide Spectrum Match 
               
            
           
           
               
               
               
               
               
            
               
                   
                 SEQ ID 
                 Injection 
                 Injection 
                 Injection 
               
               
                 Sequence 
                 NO: 
                 1 
                 2 
                 3 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 AAINKWVSnKTEGR 
                 336 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 AALAAFnAQNnGSnFQLEEISR 
                 4 
                 114 
                 120 
                 119 
               
               
                   
               
               
                 AATcINPLnGSVcERPAnHSAK 
                 5 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 ADGTVnQIEGEATPVnLTEPAK 
                 6 
                 20 
                 19 
                 19 
               
               
                   
               
               
                 ADGTVNQIEGEATPVnLTEPAKLEVK 
                 7 
                 12 
                 11 
                 13 
               
               
                   
               
               
                 ADTHDEILEGLNFnLTEIPEAQIH 
                 8 
                 10 
                 7 
                 8 
               
               
                   
               
               
                 ADTHDEILEGLnFnLTEIPEAQIHEGFQELLR 
                 9 
                 118 
                 122 
                 117 
               
               
                   
               
               
                 AELSnHTRPVILVPGcLGnQLEAK 
                 10 
                 2 
                 3 
                 2 
               
               
                   
               
               
                 AFEnVTDLQWLILDHnLLEnSK 
                 11 
                 15 
                 17 
                 18 
               
               
                   
               
               
                 AFHYnVSSHGcQLLPWTQHSPHTR 
                 12 
                 2 
                 2 
                 2 
               
               
                   
               
               
                 AFITnFSMIIDGmTYPGIIK 
                 13 
                 7 
                 7 
                 7 
               
               
                   
               
               
                 AFITnFSMIIDGmTYPGIIKEK 
                 14 
                 3 
                 3 
                 3 
               
               
                   
               
               
                 AGAFLGLTNVAVmnLSGNcLR 
                 15 
                 9 
                 10 
                 8 
               
               
                   
               
               
                 AGLQAFFQVQEcnK 
                 16 
                 5 
                 5 
                 7 
               
               
                   
               
               
                 AHLnVSGIPcSVLLADVEDLIQQQISnDTVSPR 
                 17 
                 2 
                 1 
                 1 
               
               
                   
               
               
                 ALPQPQnVTSLLGcTH 
                 18 
                 6 
                 7 
                 5 
               
               
                   
               
               
                 ALYAWNNGHQILYnVTLFHVIR 
                 337 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 AnLSSQALQmSLDYGFVTPLTSMSIR 
                 21 
                 12 
                 12 
                 13 
               
               
                   
               
               
                 APDKNVIFSPLSISTALAFLSLGAHnTTLTEILK 
                 22 
                 9 
                 9 
                 9 
               
               
                   
               
               
                 AQLLQGLGFnLTER 
                 23 
                 20 
                 19 
                 18 
               
               
                   
               
               
                 AQVIInITDVDEPPIFQQPFYHFQLK 
                 24 
                 3 
                 2 
                 2 
               
               
                   
               
               
                 AREDIFMETLKDIVEYYNDSnGSHVLQGR 
                 25 
                 21 
                 22 
                 21 
               
               
                   
               
               
                 AVLQLnEEGVDTAGSTGVTLnLTSKPIILR 
                 26 
                 34 
                 38 
                 37 
               
               
                   
               
               
                 AVnITSENLIDDVVSLIR 
                 27 
                 11 
                 12 
                 10 
               
               
                   
               
               
                 AYLLPAPPAPGnASESEEDR 
                 28 
                 3 
                 3 
                 3 
               
               
                   
               
               
                 cATPHGDnASLEATFVK 
                 29 
                 3 
                 3 
                 3 
               
               
                   
               
               
                 cGLVPVLAENYnK 
                 30 
                 2 
                 3 
                 4 
               
               
                   
               
               
                 cGncSLTTLKDEDFcK 
                 31 
                 7 
                 6 
                 7 
               
               
                   
               
               
                 cGncSLTTLKDEDFcKR 
                 32 
                 7 
                 8 
                 7 
               
               
                   
               
               
                 cIQAnYSLmENGK 
                 33 
                 15 
                 21 
                 21 
               
               
                   
               
               
                 cIQAnYSLmEnGKIK 
                 34 
                 2 
                 4 
                 2 
               
               
                   
               
               
                 cmWSSALnSLnLSFAGLEQVPK 
                 35 
                 3 
                 4 
                 3 
               
               
                   
               
               
                 cSDGWSFDATTLDDnGTmLFFK 
                 36 
                 16 
                 17 
                 19 
               
               
                   
               
               
                 DFVnASSKYEITTIHNLFR 
                 37 
                 4 
                 4 
                 3 
               
               
                   
               
               
                 DHEnGTGTNTYAALNSVYLMMNNQMR 
                 38 
                 7 
                 8 
                 6 
               
               
                   
               
               
                 DIVEYYnDSnGSHVLQGR 
                 39 
                 34 
                 36 
                 37 
               
               
                   
               
               
                 DKIcDLLVANNHFAHFFAPQnLTNmNK 
                 40 
                 34 
                 32 
                 31 
               
               
                   
               
               
                 DmTEVISSLENANYKDHEnGTGTnTYAALNSVYLMMNNQmR 
                 41 
                 7 
                 8 
                 6 
               
               
                   
               
               
                 DQcIVDDITYNVnDTFHK 
                 42 
                 11 
                 10 
                 11 
               
               
                   
               
               
                 DRQDGEEVLQcmPVcGRPVTPIAQnQTTLGSSR 
                 44 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 DSVSVVLGQHFFnR 
                 45 
                 3 
                 3 
                 3 
               
               
                   
               
               
                 DTAVFEcLPQHAmFGNDTITcTTHGnWTK 
                 46 
                 32 
                 29 
                 32 
               
               
                   
               
               
                 DTAVFEcLPQHAmFGnDTITcTTHGnWTKLPEcR 
                 47 
                 11 
                 9 
                 10 
               
               
                   
               
               
                 DVQIIVFPEDGIHGFnFTR 
                 48 
                 8 
                 8 
                 7 
               
               
                   
               
               
                 EDIFmETLKDIVEYYnDSNGSHVLQGR 
                 49 
                 4 
                 5 
                 4 
               
               
                   
               
               
                 EEQYNSTYRVVSVLTVLHQDWLnGKEYK 
                 51 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 EGDHEFLEVPEAQEDVEATFPVHQPGnYScSYR 
                 52 
                 19 
                 16 
                 17 
               
               
                   
               
               
                 EGYSnISYIVVNHQGISSR 
                 53 
                 10 
                 9 
                 9 
               
               
                   
               
               
                 EHEAQSnASLDVFLGHTNVEELmK 
                 54 
                 11 
                 10 
                 9 
               
               
                   
               
               
                 EHEGAIYPDnTTDFQR 
                 55 
                 25 
                 25 
                 26 
               
               
                   
               
               
                 EHETcLAPELYNGnYSTTQK 
                 56 
                 6 
                 7 
                 5 
               
               
                   
               
               
                 EHYnLSAATcSPGQmcGHYTQVVWAK 
                 57 
                 2 
                 3 
                 2 
               
               
                   
               
               
                 ELDREVYPWYnLTVEAK 
                 58 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 ELHHLQEQnVSNAFLDK 
                 59 
                 25 
                 28 
                 27 
               
               
                   
               
               
                 ELHHLQEQnVSnAFLDKGEFYIGSK 
                 60 
                 101 
                 100 
                 94 
               
               
                   
               
               
                 ELPGVcnETmmALWEEcKPcLK 
                 61 
                 8 
                 9 
                 10 
               
               
                   
               
               
                 EnLTAPGSDSAVFFEQGTTR 
                 62 
                 17 
                 16 
                 16 
               
               
                   
               
               
                 EQFcPPPPQIPNAQnMTTTVNYQDGEK 
                 338 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 ERSWPAVGncSSALR 
                 63 
                 2 
                 2 
                 2 
               
               
                   
               
               
                 EVnTSGFAPARPPPQPGSTTFWAWSVLR 
                 64 
                 6 
                 5 
                 5 
               
               
                   
               
               
                 EVSFLncSLDnGGcTHYcLEEVGWR 
                 65 
                 6 
                 7 
                 5 
               
               
                   
               
               
                 EVYPWYnLTVEAK 
                 66 
                 2 
                 2 
                 2 
               
               
                   
               
               
                 EWEKELHHLQEQnVSnAFLDKGEFYIGSK 
                 67 
                 6 
                 3 
                 5 
               
               
                   
               
               
                 EYESYSDFERnVTEK 
                 68 
                 2 
                 1 
                 1 
               
               
                   
               
               
                 FcRDnYTDLVAIQNK 
                 69 
                 1 
                 1 
                 2 
               
               
                   
               
               
                 FDFQGTcEYLLSAPcHGPPLGAEnFTVTVAnEHR 
                 70 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 FEDGVLDPDYPRnISDGFDGIPDnVDAALALPAHSYSGR 
                 71 
                 8 
                 7 
                 7 
               
               
                   
               
               
                 FEVDSPVYnATWSASLK 
                 72 
                 4 
                 4 
                 3 
               
               
                   
               
               
                 FGHSAVLHnSTMYVFGGFNSLLLSDILVFTSEQcDAHR 
                 73 
                 5 
                 7 
                 5 
               
               
                   
               
               
                 FHDVSESTHWTPFLnASVHYIR 
                 74 
                 6 
                 6 
                 6 
               
               
                   
               
               
                 FLNnGTcTAEGK 
                 75 
                 1 
                 1 
                 3 
               
               
                   
               
               
                 FnLTETSEAEIHQSFQHLLR 
                 78 
                 183 
                 177 
                 182 
               
               
                   
               
               
                 FNPGAESVVLSnSTLK 
                 79 
                 2 
                 2 
                 1 
               
               
                   
               
               
                 FnSSYLQGTNQITGR 
                 80 
                 3 
                 1 
                 2 
               
               
                   
               
               
                 FQSPAGTEALFELHNISVADSAnYScVYVDLKPPFGGSAPSER 
                 81 
                 87 
                 85 
                 93 
               
               
                   
               
               
                 FSDGLESnSSTQFEVK 
                 82 
                 40 
                 44 
                 52 
               
               
                   
               
               
                 FSDGLESnSSTQFEVKK 
                 83 
                 1 
                 3 
                 3 
               
               
                   
               
               
                 FSLLGHASIScTVEnETIGVWRPSPPTcEK 
                 84 
                 68 
                 71 
                 73 
               
               
                   
               
               
                 FSYSKnETYQLFLSYSSK 
                 85 
                 8 
                 8 
                 10 
               
               
                   
               
               
                 FVGTPEVnQTTLYQR 
                 86 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 FVQAIcEGDDcQPPAYTYNnITcASPPEVVGLDLR 
                 87 
                 6 
                 4 
                 6 
               
               
                   
               
               
                 FVQGnSTEVAcHPGYGLPK 
                 88 
                 2 
                 3 
                 4 
               
               
                   
               
               
                 GAFISnFSmTVDGK 
                 89 
                 11 
                 10 
                 9 
               
               
                   
               
               
                 GcnDSDVLAVAGFALR 
                 90 
                 3 
                 4 
                 3 
               
               
                   
               
               
                 GcScFSDWQGPGcSVPVPAnQSFWTR 
                 91 
                 3 
                 5 
                 4 
               
               
                   
               
               
                 GcVLLSYLnETVTVSASLESVR 
                 92 
                 124 
                 135 
                 132 
               
               
                   
               
               
                 GDSGGPLVcmDAnnVTYVWGVVSWGEncGKPEFPGVYTK 
                 93 
                 14 
                 14 
                 12 
               
               
                   
               
               
                 GELnTSIFSSRPIDK 
                 339 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 GETHEQVHSILHFKDFVnASSK 
                 94 
                 1 
                 3 
                 1 
               
               
                   
               
               
                 GETHEQVHSILHFKDFVnASSKYEITTIHNLFR 
                 95 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 GFGVAIVGnYTAALPTEAALR 
                 96 
                 46 
                 47 
                 49 
               
               
                   
               
               
                 GFLALYQTVAVnYSQPISEASR 
                 97 
                 6 
                 7 
                 7 
               
               
                   
               
               
                 GFYPSDIAVEWESSGQPEnnYnTTPPmLDSDGSFFLYSK 
                 340 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 GGETAQSADPQWEQLnnKnLSmPLLPADFHK 
                 98 
                 12 
                 10 
                 13 
               
               
                   
               
               
                 GGNSnGALcHFPFLYNnHnYTDcTSEGR 
                 99 
                 14 
                 16 
                 14 
               
               
                   
               
               
                 GGNSNGALcHFPFLYnNHnYTDcTSEGRR 
                 100 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 GLKFnLTETSEAEIHQSFQHLLR 
                 102 
                 85 
                 79 
                 86 
               
               
                   
               
               
                 GLTFQQnASSmcGPDQDTAIR 
                 104 
                 9 
                 10 
                 8 
               
               
                   
               
               
                 GLTFQQnASSmcVPDQDTAIR 
                 105 
                 9 
                 9 
                 9 
               
               
                   
               
               
                 GmnLTVFGGTVTAFLGIPYAQPPLGR 
                 106 
                 6 
                 5 
                 4 
               
               
                   
               
               
                 GNEANYYSNATTDEHGLVQFSInTTnVmGTSLTVR 
                 107 
                 143 
                 138 
                 134 
               
               
                   
               
               
                 GNVAVTVSGHTcQHWSAQTPHTHnR 
                 108 
                 6 
                 7 
                 9 
               
               
                   
               
               
                 GSFPWQAKMVSHHnLTTGATLINEQWLLTTAK 
                 341 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 GTAnTTTAGVPcQR 
                 110 
                 3 
                 3 
                 2 
               
               
                   
               
               
                 GTGnDTVLNVALLNVISNQEcNIK 
                 111 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 GVTSVSQIFHSPDLAIRDTFVnASR 
                 112 
                 5 
                 5 
                 3 
               
               
                   
               
               
                 HAnWTLTPLK 
                 113 
                 5 
                 4 
                 5 
               
               
                   
               
               
                 HEEGHmLncTcFGQGR 
                 114 
                 6 
                 7 
                 6 
               
               
                   
               
               
                 HGIQYFnnNTQHSSLFmLNEVK 
                 115 
                 25 
                 22 
                 29 
               
               
                   
               
               
                 HGIQYFnnNTQHSSLFmLNEVKR 
                 116 
                 12 
                 14 
                 14 
               
               
                   
               
               
                 HGIQYFnnNTQHSSLFTLnEVK 
                 117 
                 39 
                 39 
                 40 
               
               
                   
               
               
                 HGIQYFnnNTQHSSLFTLNEVKR 
                 118 
                 10 
                 10 
                 11 
               
               
                   
               
               
                 HGVIISSTVDTYEnGSSVEYR 
                 119 
                 9 
                 11 
                 10 
               
               
                   
               
               
                 HLQmDIHIFEPQGISFLETESTFmTNQLVDALTTWQnK 
                 120 
                 6 
                 8 
                 5 
               
               
                   
               
               
                 HSHNNnSSDLHPHK 
                 342 
                 1 
                 4 
                 #N/A 
               
               
                   
               
               
                 HYLVSnISHDTVLQcHFTcSGK 
                 121 
                 2 
                 2 
                 3 
               
               
                   
               
               
                 HYTnSSQDVTVPcR 
                 122 
                 14 
                 13 
                 13 
               
               
                   
               
               
                 HYYIAAEEIIWnYAPSGIDIFTKEnLTAPGSDSAVFFEQGTTR 
                 123 
                 8 
                 8 
                 11 
               
               
                   
               
               
                 IADAHLDRVEnTTVYYLVLDVQESDcSVLSR 
                 124 
                 65 
                 68 
                 68 
               
               
                   
               
               
                 IcDLLVANNHFAHFFAPQnLTnMNK 
                 125 
                 25 
                 21 
                 23 
               
               
                   
               
               
                 IDSTGnVTNELR 
                 126 
                 1 
                 2 
                 3 
               
               
                   
               
               
                 IITILEEEmnVSVcGLYTYGKPVPGHVIVSIcR 
                 127 
                 79 
                 82 
                 74 
               
               
                   
               
               
                 INNDFNYEFYnSTWSYVK 
                 128 
                 6 
                 6 
                 6 
               
               
                   
               
               
                 IPcSQPPQIEHGTInSSR 
                 129 
                 20 
                 20 
                 21 
               
               
                   
               
               
                 ISEEnETTcYMGK 
                 130 
                 14 
                 15 
                 14 
               
               
                   
               
               
                 ISnSSDTVEcEcSENWK 
                 131 
                 4 
                 5 
                 5 
               
               
                   
               
               
                 ISnSSDTVEcEcSEnWKGEAcDIPHcTDncGFPHR 
                 132 
                 5 
                 4 
                 3 
               
               
                   
               
               
                 ITPNLAEFAFSLYRQLAHQSnSTNIFFSPVSIATAFAmLSLGTK 
                 133 
                 2 
                 1 
                 2 
               
               
                   
               
               
                 ITYSIVQTncSK 
                 134 
                 5 
                 8 
                 12 
               
               
                   
               
               
                 ITYSIVQTncSKEnFLFLTPDcK 
                 135 
                 11 
                 9 
                 11 
               
               
                   
               
               
                 IVGGTnSSWGEWPWQVSLQVK 
                 136 
                 8 
                 8 
                 8 
               
               
                   
               
               
                 IVLDPSGSMnIYLVLDGSDSIGASnFTGAK 
                 137 
                 135 
                 132 
                 134 
               
               
                   
               
               
                 IYPGVDFGGEELnVTFVK 
                 138 
                 5 
                 7 
                 7 
               
               
                   
               
               
                 IYSGILnLSDITK 
                 139 
                 6 
                 7 
                 7 
               
               
                   
               
               
                 lYSnHSALESLALIPLQAPLK 
                 140 
                 4 
                 3 
                 3 
               
               
                   
               
               
                 KAFITnFSMIIDGmTYPGIIK 
                 141 
                 4 
                 4 
                 4 
               
               
                   
               
               
                 KAFITnFSMIIDGmTYPGIIKEK 
                 142 
                 6 
                 6 
                 4 
               
               
                   
               
               
                 KcGncSLTTLKDEDFcK 
                 143 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 KDFEDLYTPVnGSIVIVR 
                 144 
                 2 
                 1 
                 2 
               
               
                   
               
               
                 KEDALnETR 
                 343 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 KEHETcLAPELYNGnYSTTQK 
                 145 
                 10 
                 11 
                 12 
               
               
                   
               
               
                 KIVLDPSGSMnIYLVLDGSDSIGASnFTGAK 
                 146 
                 36 
                 49 
                 42 
               
               
                   
               
               
                 KLHINHNnLTESVGPLPK 
                 147 
                 8 
                 8 
                 6 
               
               
                   
               
               
                 KLINDYVKnGTR 
                 148 
                 2 
                 2 
                 2 
               
               
                   
               
               
                 KLPPGLLAnFTLLR 
                 149 
                 4 
                 5 
                 5 
               
               
                   
               
               
                 KLSSWVLLmKYLGnATAIFFLPDEGK 
                 344 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 KnQSVNVFLGHTAIDEMLK 
                 150 
                 6 
                 4 
                 3 
               
               
                   
               
               
                 KQVHFFVnASDVDNVK 
                 151 
                 6 
                 6 
                 5 
               
               
                   
               
               
                 KVcQDcPLLAPLnDTR 
                 152 
                 14 
                 14 
                 12 
               
               
                   
               
               
                 LAGKPTHVnVSVVMAEVDGTcY 
                 153 
                 54 
                 56 
                 64 
               
               
                   
               
               
                 LAnLTQGEDQYYLR 
                 154 
                 14 
                 15 
                 15 
               
               
                   
               
               
                 LATALSLSNKFVEGSHnSTVSLTTK 
                 155 
                 2 
                 2 
                 2 
               
               
                   
               
               
                 LDAPTNLQFVnETDSTVLVR 
                 156 
                 9 
                 8 
                 9 
               
               
                   
               
               
                 LDPVSLQTLQTWnTSYPK 
                 157 
                 2 
                 2 
                 1 
               
               
                   
               
               
                 LDREnISEYHLTAVIVDK 
                 158 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 LDREnISEYHLTAVIVDKDTGEnLETPSSFTIK 
                 159 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 LEDLEVTGSSFLnLSTnIFSnLTSLGK 
                 160 
                 8 
                 10 
                 12 
               
               
                   
               
               
                 LEPVHLQLQcMSQEQLAQVAAnATK 
                 161 
                 11 
                 12 
                 12 
               
               
                   
               
               
                 LETTVnYTDSQRPIcLPSK 
                 162 
                 3 
                 2 
                 3 
               
               
                   
               
               
                 LFGDKSLTFnETYQDISELVYGAK 
                 163 
                 5 
                 6 
                 4 
               
               
                   
               
               
                 LGAcnDTLQQLMEVFK 
                 164 
                 32 
                 31 
                 34 
               
               
                   
               
               
                 LGAcnDTLQQLmEVFKFDTISEK 
                 165 
                 10 
                 10 
                 9 
               
               
                   
               
               
                 LGAcnDTLQQLMEVFKFDTISEKTSDQIHFFFAK 
                 166 
                 4 
                 4 
                 3 
               
               
                   
               
               
                 LGHcPDPVLVnGEFSSSGPVnVSDK 
                 167 
                 8 
                 9 
                 9 
               
               
                   
               
               
                 LGSFEGLVnLTFIHLQHNR 
                 168 
                 9 
                 11 
                 13 
               
               
                   
               
               
                 LGSLQELFLDSnnISELPPQVFSQLFcLER 
                 169 
                 4 
                 2 
                 3 
               
               
                   
               
               
                 LGSYPVGGnVSFEcEDGFILR 
                 170 
                 6 
                 6 
                 5 
               
               
                   
               
               
                 LGTSLSSGHVLMnGTLK 
                 171 
                 8 
                 8 
                 8 
               
               
                   
               
               
                 LHINHNnLTESVGPLPK 
                 172 
                 10 
                 14 
                 11 
               
               
                   
               
               
                 LKELPGVcnETMmALWEEcKPcLK 
                 173 
                 14 
                 12 
                 11 
               
               
                   
               
               
                 LLLSQLDSHPSHSAVVnWTSYASSIEALSSGNK 
                 174 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 LNAENnATFYFK 
                 175 
                 75 
                 103 
                 108 
               
               
                   
               
               
                 LnDTLDYEcHDGYESnTGSTTGSIVcGYnGWSDLPIcYER 
                 176 
                 17 
                 19 
                 14 
               
               
                   
               
               
                 LNVEAAnWTVR 
                 177 
                 4 
                 3 
                 4 
               
               
                   
               
               
                 LPPGLLAnFTLLR 
                 179 
                 4 
                 4 
                 3 
               
               
                   
               
               
                 LPTQnITFQTESSVAEQEAEFQSPK 
                 180 
                 28 
                 28 
                 30 
               
               
                   
               
               
                 LPYQGnATmLVVLmEK 
                 181 
                 1 
                 2 
                 2 
               
               
                   
               
               
                 LQAILGVPWKDKncTSR 
                 182 
                 12 
                 13 
                 13 
               
               
                   
               
               
                 LQAPLnYTEFQKPIcLPSK 
                 183 
                 7 
                 7 
                 7 
               
               
                   
               
               
                 LQNnENnIScVER 
                 184 
                 6 
                 7 
                 7 
               
               
                   
               
               
                 LSDLSInSTEcLHVHcR 
                 185 
                 81 
                 100 
                 84 
               
               
                   
               
               
                 LSHnELADSGIPGnSFnVSSLVELDLSYNK 
                 186 
                 19 
                 23 
                 26 
               
               
                   
               
               
                 LSLHRPALEDLLLGSEAnLTcTLTGLR 
                 187 
                 88 
                 88 
                 92 
               
               
                   
               
               
                 LSSWVLLmKYLGnATAIFFLPDEGK 
                 188 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 LSVDKDQYVEPEnVTIQcDSGYGVVGPQSITcSGnR 
                 189 
                 4 
                 4 
                 5 
               
               
                   
               
               
                 LTDTIcGVGnmSAnASDQER 
                 190 
                 3 
                 5 
                 5 
               
               
                   
               
               
                 LVSANRLFGDKSLTFnETYQDISELVYGAK 
                 191 
                 2 
                 3 
                 3 
               
               
                   
               
               
                 LYHFLLGAWSLnATELDPcPLSPELLGLTK 
                 192 
                 19 
                 20 
                 15 
               
               
                   
               
               
                 LYLGSNnLTALHPALFQnLSK 
                 193 
                 11 
                 10 
                 12 
               
               
                   
               
               
                 mAGKPTHInVSVVmAEADGTcY 
                 194 
                 3 
                 4 
                 2 
               
               
                   
               
               
                 mAGKPTHVnVSVVmAEVDGTcY 
                 195 
                 4 
                 3 
                 3 
               
               
                   
               
               
                 mAWPEDHVFISTPSFnYTGR 
                 196 
                 4 
                 4 
                 6 
               
               
                   
               
               
                 MDGASnVTcInSR 
                 197 
                 23 
                 24 
                 25 
               
               
                   
               
               
                 MLLTFHTDFSNEEnGTImFYK 
                 198 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 mLnTSSLLEQLnEQFNWVSR 
                 200 
                 27 
                 27 
                 26 
               
               
                   
               
               
                 mPSQAPTGNFYPQPLLnSSmcLEDSR 
                 201 
                 2 
                 4 
                 4 
               
               
                   
               
               
                 mVSHHnLTTGATLInEQWLLTTAK 
                 204 
                 453 
                 443 
                 443 
               
               
                   
               
               
                 MVSHHnLTTGATLInEQWLLTTAKNLFLnHSEnATAK 
                 205 
                 12 
                 11 
                 10 
               
               
                   
               
               
                 mVTAFTTccTLSEEFAcVDNLADLVFGELcGVNEnR 
                 206 
                 2 
                 3 
                 2 
               
               
                   
               
               
                 NAHGEEKEnLTAR 
                 207 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 NcGVncSGDVFTALIGEIASPnYPKPYPEnSR 
                 208 
                 7 
                 8 
                 8 
               
               
                   
               
               
                 NEEYnKSVQEIQATFFYFTPnKTEDTIFLR 
                 210 
                 11 
                 11 
                 12 
               
               
                   
               
               
                 nEMLEIQVFNYSKVFSnK 
                 211 
                 2 
                 1 
                 2 
               
               
                   
               
               
                 nGTGHGnSTHHGPEYmR 
                 212 
                 4 
                 6 
                 2 
               
               
                   
               
               
                 NHPnITFFVYVSnFTWPIK 
                 213 
                 3 
                 4 
                 4 
               
               
                   
               
               
                 nISDGFDGIPDNVDAALALPAHSYSGR 
                 214 
                 2 
                 2 
                 2 
               
               
                   
               
               
                 NLASRPYTFHSHGITYYKEHEGAIYPDnTTDFQR 
                 215 
                 5 
                 4 
                 5 
               
               
                   
               
               
                 NLFLnHSENATAK 
                 216 
                 248 
                 255 
                 243 
               
               
                   
               
               
                 NLFLnHSEnATAKDIAPTLTLYVGK 
                 217 
                 9 
                 11 
                 11 
               
               
                   
               
               
                 NLFLnHSEnATAKDIAPTLTLYVGKK 
                 218 
                 2 
                 3 
                 2 
               
               
                   
               
               
                 NmASRPYSIYPHGVTFSPYEDEVnSSFTSGR 
                 345 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 nnATVHEQVGGPSLTSDLQAQSK 
                 219 
                 41 
                 45 
                 51 
               
               
                   
               
               
                 NnmSFVVLVPTHFEWnVSQVLAnLSWDTLHPPLVWERPTK 
                 220 
                 2 
                 1 
                 2 
               
               
                   
               
               
                 NPPmGGNVVIFDTVITnQEEPYQnHSGR 
                 221 
                 6 
                 5 
                 5 
               
               
                   
               
               
                 NPVGLIGAEnATGETDPSHSK 
                 222 
                 10 
                 10 
                 11 
               
               
                   
               
               
                 nQALnLSLAYSFVTPLTSMVVTKPDDQEQSQVAEKPmEGESR 
                 223 
                 8 
                 9 
                 9 
               
               
                   
               
               
                 NSVLnSSTAEHSSPYSEDPIEDPLQPDVTGIR 
                 224 
                 2 
                 4 
                 3 
               
               
                   
               
               
                 NVIFSPLSISTALAFLSLGAHnTTLTEILK 
                 225 
                 9 
                 10 
                 11 
               
               
                   
               
               
                 QDQcIYnTTYLnVQR 
                 226 
                 160 
                 164 
                 181 
               
               
                   
               
               
                 QDQcIYnTTYLNVQREnGTISR 
                 227 
                 5 
                 4 
                 5 
               
               
                   
               
               
                 QEDLSVGSVLLTVnATDPDSLQHQTIR 
                 228 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 QGGVnATQVLIQHLR 
                 229 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 QInSSISGNLWDKDQR 
                 230 
                 1 
                 1 
                 3 
               
               
                   
               
               
                 QLAHQSnSTnIFFSPVSIATAFAMLSLGTK 
                 231 
                 83 
                 86 
                 92 
               
               
                   
               
               
                 QLDmLDLSnNSLASVPEGLWASLGQPnWDmR 
                 232 
                 18 
                 15 
                 16 
               
               
                   
               
               
                 QLEEFLnQSSPFYFWmnGDR 
                 233 
                 28 
                 27 
                 27 
               
               
                   
               
               
                 QLEEFLnQSSPFYFWmnGDRIDSLLEnDR 
                 234 
                 11 
                 11 
                 11 
               
               
                   
               
               
                 QLVEIEKVVLHPnYSQVDIGLIK 
                 235 
                 6 
                 5 
                 5 
               
               
                   
               
               
                 QNESHnFSGDIALLELQHSIPLGPNVLPVcLPDnETLYR 
                 236 
                 5 
                 6 
                 5 
               
               
                   
               
               
                 QnQcFYnSSYLnVQR 
                 237 
                 17 
                 18 
                 18 
               
               
                   
               
               
                 QPQAGLSQAnFTLGPVSR 
                 238 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 QQQHLFGSnVTDcSGNFcLFR 
                 239 
                 159 
                 161 
                 160 
               
               
                   
               
               
                 QVHFFVnASDVDNVK 
                 240 
                 12 
                 11 
                 12 
               
               
                   
               
               
                 QVLFLDTVYGncSTHFTVK 
                 241 
                 5 
                 4 
                 4 
               
               
                   
               
               
                 QVQVLQnLTTTYEIVLWQPVTADLIVK 
                 242 
                 2 
                 3 
                 2 
               
               
                   
               
               
                 REGDHEFLEVPEAQEDVEATFPVHQPGnYScSYR 
                 243 
                 12 
                 14 
                 12 
               
               
                   
               
               
                 RHEEGHmLncTcFGQGR 
                 244 
                 3 
                 4 
                 1 
               
               
                   
               
               
                 RNPPmGGNVVIFDTVITnQEEPYQnHSGR 
                 245 
                 13 
                 12 
                 12 
               
               
                   
               
               
                 SDHGSSIScQPPAEIPGYLPADTVHLAVEFFNLTHLPAnLLQGASK 
                 246 
                 9 
                 9 
                 10 
               
               
                   
               
               
                 SHAASDAPEnLTLLAETADAR 
                 247 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 SHEIWTHScPQSPGnGTDASH 
                 248 
                 1 
                 2 
                 2 
               
               
                   
               
               
                 SIPAcVPWSPYLFQPnDTcIVSGWGR 
                 249 
                 14 
                 12 
                 13 
               
               
                   
               
               
                 SKPTVSSSmEFKYDFnSSmLYSTAK 
                 250 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 SKWnITmESYVVHTNYDEYAIFLTK 
                 251 
                 19 
                 18 
                 19 
               
               
                   
               
               
                 SLGnVnFTVSAEALESQELcGTEVPSVPEHGR 
                 252 
                 146 
                 155 
                 157 
               
               
                   
               
               
                 SLGnVnFTVSAEALESQELcGTEVPSVPEHGRK 
                 253 
                 3 
                 3 
                 3 
               
               
                   
               
               
                 SLTFnETYQDISELVYGAK 
                 254 
                 34 
                 28 
                 29 
               
               
                   
               
               
                 SPYEMFGDEEVmcLNGnWTEPPQcK 
                 255 
                 28 
                 30 
                 31 
               
               
                   
               
               
                 SPYYnVSDEISFHcYDGYTLR 
                 256 
                 153 
                 152 
                 157 
               
               
                   
               
               
                 SQILEGLGFnLTELSESDVHR 
                 257 
                 20 
                 21 
                 20 
               
               
                   
               
               
                 SRVYLQGLIDcYLFGnSSTVLEDSK 
                 258 
                 2 
                 2 
                 1 
               
               
                   
               
               
                 SRYPHKPEInSTTHPGADLQENFcR 
                 259 
                 13 
                 14 
                 16 
               
               
                   
               
               
                 STGKPTLYnVSLVMSDTAGTcY 
                 261 
                 7 
                 6 
                 6 
               
               
                   
               
               
                 SVQEIQATFFYFTPnKTEDTIFLR 
                 262 
                 95 
                 96 
                 104 
               
               
                   
               
               
                 SVTLQIYnHSLTLSAR 
                 346 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 SWPAVGncSSALR 
                 347 
                 1 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 TEGRPDmKTELFSSScPGGImLnETGQGYQR 
                 264 
                 3 
                 2 
                 2 
               
               
                   
               
               
                 TELFSSScPGGImLnETGQGYQR 
                 265 
                 11 
                 13 
                 12 
               
               
                   
               
               
                 TEVSSnHVLIYLDKVSnQTLSLFFTVLQDVPVR 
                 266 
                 7 
                 8 
                 7 
               
               
                   
               
               
                 TEVSSnHVLIYLDKVSnQTLSLFFTVLQDVPVRDLKPAIVK 
                 267 
                 3 
                 3 
                 2 
               
               
                   
               
               
                 THTnISESHPnATFSAVGEASIcEDDWnSGER 
                 268 
                 17 
                 16 
                 20 
               
               
                   
               
               
                 TKPREEQYnSTYR 
                 269 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 TLFcnASKEWDnTTTEcR 
                 270 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 TLnQSSDELQLSmGnAmFVK 
                 271 
                 184 
                 190 
                 206 
               
               
                   
               
               
                 TLYETEVFSTDFSnISAAK 
                 272 
                 8 
                 9 
                 9 
               
               
                   
               
               
                 TTTVQVPmMHQmEQYYHLVDmELncTVLQMDYSK 
                 273 
                 8 
                 8 
                 10 
               
               
                   
               
               
                 TVIRPFYLTnSSGVD 
                 274 
                 3 
                 2 
                 3 
               
               
                   
               
               
                 TVLTPATNHmGnVTFTIPAnR 
                 275 
                 25 
                 22 
                 26 
               
               
                   
               
               
                 TVLTPATNHmGnVTFTIPAnREFK 
                 276 
                 2 
                 3 
                 2 
               
               
                   
               
               
                 TVVTYHIPQnSSLENVDSR 
                 277 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 TYnVLDmKnTTcQDLQIEVTVK 
                 278 
                 3 
                 3 
                 7 
               
               
                   
               
               
                 VASVININPnTTHSTGScR 
                 279 
                 2 
                 3 
                 3 
               
               
                   
               
               
                 VcQDcPLLAPLnDTR 
                 280 
                 24 
                 29 
                 25 
               
               
                   
               
               
                 VcQDcPLLAPLnDTRVVHAAK 
                 281 
                 6 
                 6 
                 6 
               
               
                   
               
               
                 VDKDLQSLEDILHQVEnK 
                 282 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 VEGSSSHLVTFTVLPLEIGLHNInFSLETWFGK 
                 283 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 VEnTTVYYLVLDVQESDcSVLSR 
                 284 
                 23 
                 24 
                 22 
               
               
                   
               
               
                 VFHIHnESWVLLTPK 
                 285 
                 2 
                 3 
                 4 
               
               
                   
               
               
                 VFPLSLDSTPQDGNVVVAcLVQGFFPQEPLSVTWSESGQnVTAR 
                 286 
                 13 
                 15 
                 12 
               
               
                   
               
               
                 VGQLQLSHnLSLVILVPQNLK 
                 287 
                 19 
                 19 
                 19 
               
               
                   
               
               
                 VIDFncTTSSVSSALAnTK 
                 288 
                 18 
                 18 
                 18 
               
               
                   
               
               
                 VIDFncTTSSVSSALAnTKDSPVLIDFFEDTER 
                 289 
                 2 
                 1 
                 1 
               
               
                   
               
               
                 VKPnPPHNLSVINSEELSSILK 
                 348 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 VLSNNSDAnLELINTWVAK 
                 290 
                 51 
                 50 
                 54 
               
               
                   
               
               
                 VLTLNLDQVDFQHAGnYScVASNVQGK 
                 291 
                 2 
                 #N/A 
                 2 
               
               
                   
               
               
                 VLYLAAYncTLRPVSK 
                 292 
                 8 
                 8 
                 7 
               
               
                   
               
               
                 VPGnVTAVLGETLK 
                 293 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 VPMMLQSSTISYLHDSELPcQLVQMnYVGnGTVFFILPDK 
                 294 
                 11 
                 9 
                 7 
               
               
                   
               
               
                 VPMMLQSSTISYLHDSELPcQLVQMNYVGnGTVFFILPDKGK 
                 349 
                 2 
                 #N/A 
                 #N/A 
               
               
                   
               
               
                 VSAITLVSATSTTAnmTVGPEGK 
                 295 
                 3 
                 3 
                 4 
               
               
                   
               
               
                 VSEHIPVYQQEEnQTDVWTLLNGSK 
                 296 
                 7 
                 7 
                 8 
               
               
                   
               
               
                 VSEHIPVYQQEEnQTDVWTLLnGSKDDFLIYDR 
                 297 
                 8 
                 7 
                 6 
               
               
                   
               
               
                 VSLTnVSISDEGR 
                 298 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 VSnQTLSLFFTVLQDVPVR 
                 299 
                 30 
                 27 
                 28 
               
               
                   
               
               
                 VSnQTLSLFFTVLQDVPVRDLKPAIVK 
                 350 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 VSnVScQASVSR 
                 300 
                 1 
                 2 
                 1 
               
               
                   
               
               
                 VSTVYANnGSVLQGTSVASVYHGK 
                 301 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 VTAcHSSQPnATLYK 
                 302 
                 8 
                 8 
                 7 
               
               
                   
               
               
                 VTISGVYDLGDVLEEmGIADLFTNQAnFSR 
                 303 
                 14 
                 15 
                 12 
               
               
                   
               
               
                 VTQnLTLIEESLTSEFIHDIDR 
                 304 
                 9 
                 9 
                 9 
               
               
                   
               
               
                 VTQVYAEnGTVLQGSTVASVYK 
                 305 
                 12 
                 15 
                 14 
               
               
                   
               
               
                 VTQVYAEnGTVLQGSTVASVYKGK 
                 306 
                 2 
                 3 
                 3 
               
               
                   
               
               
                 VTWKPQGAPVEWEEETVTnHTLR 
                 307 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 VVLHPnYSQVDIGLIK 
                 308 
                 32 
                 29 
                 29 
               
               
                   
               
               
                 VVLHPnYSQVDIGLIKLK 
                 309 
                 1 
                 1 
                 1 
               
               
                   
               
               
                 VYIHPFHLVIHnESTcEQLAK 
                 310 
                 11 
                 13 
                 14 
               
               
                   
               
               
                 VYKPSAGnNSLYR 
                 311 
                 6 
                 7 
                 9 
               
               
                   
               
               
                 VYLQGLIDcYLFGnSSTVLEDSK 
                 312 
                 7 
                 8 
                 5 
               
               
                   
               
               
                 VYSGILnQSEIK 
                 351 
                 1 
                 1 
                 #N/A 
               
               
                   
               
               
                 WDPEVncSmAQIQLcPPPPQIPnSHnMTTTLNYR 
                 314 
                 31 
                 31 
                 30 
               
               
                   
               
               
                 WFSAGLASnSSWLR 
                 315 
                 3 
                 3 
                 2 
               
               
                   
               
               
                 WFYIASAFRNEEYnK 
                 316 
                 2 
                 3 
                 3 
               
               
                   
               
               
                 WnITmESYVVHTNYDEYAIFLTK 
                 317 
                 12 
                 14 
                 12 
               
               
                   
               
               
                 WNPcLEPHRFnDTEVLQR 
                 352 
                 2 
                 1 
                 #N/A 
               
               
                   
               
               
                 WNVNAPPTFHSEMMYDnFTLVPVWGK 
                 318 
                 6 
                 5 
                 3 
               
               
                   
               
               
                 WVLTAAHcLLYPPWDKnFTEnDLLVR 
                 319 
                 14 
                 15 
                 14 
               
               
                   
               
               
                 YAEDKFnETTEK 
                 320 
                 5 
                 6 
                 7 
               
               
                   
               
               
                 YFYnGTSmAcETFQYGGcmGnGNNFVTEK 
                 321 
                 21 
                 26 
                 25 
               
               
                   
               
               
                 YGNPNETQnnSTSWPVFK 
                 322 
                 3 
                 4 
                 4 
               
               
                   
               
               
                 YKGLnLTEDTYKPR 
                 323 
                 2 
                 2 
                 2 
               
               
                   
               
               
                 YLGnATAIFFLPDEGK 
                 324 
                 102 
                 106 
                 115 
               
               
                   
               
               
                 YLGnATAIFFLPDEGKLQHLEnELTHDIITK 
                 325 
                 58 
                 60 
                 58 
               
               
                   
               
               
                 YLHTAVIVSGTMLVFGGnTHnDTSmSHGAK 
                 326 
                 2 
                 3 
                 2 
               
               
                   
               
               
                 YnSQNQSnNQFVLYR 
                 327 
                 10 
                 8 
                 14 
               
               
                   
               
               
                 YnWSFIHcPAcQcNGHSK 
                 328 
                 2 
                 4 
                 3 
               
               
                   
               
               
                 YPHKPEInSTTHPGADLQENFcR 
                 329 
                 16 
                 17 
                 17 
               
               
                   
               
               
                 YPPTVSmVEGQGEKnVTFWGRPLPR 
                 330 
                 1 
                 #N/A 
                 1 
               
               
                   
               
               
                 YQFNTNVVFSNnGTLVDR 
                 331 
                 8 
                 6 
                 9 
               
               
                   
               
               
                 YTcEEPYYYmEnGGGGEYHcAGnGSWVnEVLGPELPK 
                 332 
                 11 
                 12 
                 12 
               
               
                   
               
               
                 YTGnASALFILPDQDK 
                 333 
                 17 
                 16 
                 16 
               
               
                   
               
               
                 YTGnASALFILPDQDKmEEVEAmLLPETLK 
                 334 
                 26 
                 27 
                 25 
               
               
                   
               
               
                 YTGnASALFILPDQDKmEEVEAmLLPETLKR 
                 335 
                 31 
                 33 
                 31 
               
               
                   
               
               
                 YTTFEYPnTINFScNTGFYLNGADSAK 
                 353 
                 2 
                 #N/A 
                 #N/A 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Reproducibility of Glycopeptide  
               
               
                 Isolations Using a Hydrazide Tip 
               
            
           
           
               
               
            
               
                   
                 Mean CV (%) 
               
            
           
           
               
               
               
            
               
                   
                 Between  
                 Between  
               
               
                 No. of  
                 Injections  
                 Isolations 
               
               
                 Total PSMs 
                 (n = 3) 
                 (n = 3) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 PSM &gt;= 150 
                 4.53 
                 6.32 
               
               
                 150 &gt; PSM &gt;= 60 
                 6.27 
                 11.36 
               
               
                 60 &gt; PSM &gt;= 30 
                 8.57 
                 9.98 
               
               
                 30 &gt; PSM &gt;= 15 
                 11.53 
                 17.01 
               
               
                 15 &gt; PSM 
                 21.55 
                 28.1 
               
               
                   
               
            
           
         
       
     
     Peptide Isolation by Conjugation to Amino-Linking Beads 
     Several different glycoproteins were conjugated to amino-linking beads, the proteins were digested into peptides using the presently disclosed methods with amino-reactive tips and the peptides were used for global proteomics analysis. 
     Specifically, for the tube samples, casein was coupled to amino-linking beads at pH 10 for 4 h, reduced with NaCNBH 4  at pH 7 for 4 h, and the reaction sites on the beads were blocked with 1M Tris-HCl at pH 7 in the presence of NaCNBH 4  for 30 min. Then, the beads were denatured with 8M urea, reduced with TCEP, alkylated with IAA and digested with trypsin overnight. 
     Table 5 shows that conjugation of the amino-linking beads to the protein was most effective at pH 10. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Efficiency of Amino-linking Beads at Different pH  
               
               
                 Values and Capacity 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Protein  
                   
                   
                   
               
               
                   
                 Loading (μg) 
                   
                 Protein Conc.  
                   
               
               
                   
                 Per 50 μL 
                 Volume 
                 (mg/mL) 
                 % 
               
            
           
           
               
               
               
               
               
               
            
               
                 pH 
                 beads 
                 (μL) 
                 Before 
                 After 
                 Coupled 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 10 
                 ~200 
                 400 
                 0.571 
                 undetectable 
                 100 
               
               
                   
                 ~350 
                 400 
                 0.879 
                 undetectable 
                 100 
               
               
                   
                 ~680 
                 400 
                 1.703 
                 0.331 
                 80.56 
               
               
                 7 
                 ~200 
                 400 
                 0.46 
                 0.204 
                 55.65 
               
               
                   
               
            
           
         
       
     
     Example 2 
     Tissue Proteomics by Mass Spectrometry: Elimination of OCT Interference Using Chemical Immobilization of Proteins for Peptide Extraction 
     Tissue proteomics are important for the identification of disease biomarkers, treatment targets and help in the understanding of the pathological characteristics of tissues. Tissues are commonly stored in an embedding medium like optimal cutting temperature compound (OCT) in the freezer or formalin-fixed and paraffin-embedded (FFPE) at room temperature in order to maintain the tissue morphology for histology evaluation. Currently, most of the tissue proteomic studies are performed on frozen tissues or FFPE embedded tissues. Due to the malicious effect of OCT to the mass spectrometer, only a handful of proteomics studies have been performed on OCT embedded tissues (Asomugha et al.; Somiari et al., 2003; Nirmalan et al.; Palmer-Toy et al., 2005; Scicchitano et al., 2009). OCT embedded tissues are studied using either two-dimensional gel electrophoresis (2D DIGE) technology or shot gun proteomics using LC-MS/MS. 2D DIGE could separate proteins from OCT; however, most of the LC-MS/MS studies of OCT embedded tissue had OCT contamination resulting in fewer protein identifications (Nirmalan et al.; Palmer-Toy et al., 2005; Scicchitano et al., 2009). 
     Tissue proteins play important roles in biological processes. Quantitative analysis of tissue proteins and their modifications such as phosphorylation, glycosylation, acetylation, is the key to the understanding of molecular mechanism that differentiates between normal and disease states. The disease-specific proteins from tissues can also be used as biomarkers for the diagnosis of diseases or as new drug targets for drug development as therapeutics (Zhang et al., 2007). In the diseased state, tissue secretes or sheds disease-specific proteins into the body fluids such as serum, which can be used as biomarkers. However, the excreted proteins from a diseased tissue have higher concentration at the tissue site and become diluted by mixing with other proteins from other tissues in serum (Zhang et al., 2007; Li et al., 2008). An example was shown in the process of detecting prostate cancer proteins in serum using TOF/TOF (Tian et al., 2008). 
     Traditionally, tissue proteins are analyzed using immunoassays, which rely on the development of high quality antibodies. Advances in mass spectrometry (MS) and high performance liquid chromatography (HPLC) systems have led to the blossoming of proteomics (Bantscheff et al., 2007). Increases in sensitivity, resolution, and speed of the mass spectrometers have led to the rapid identification of large numbers of proteins with high confidence, making the analysis of complex samples such as tissue possible. Tissue proteome, located at the primary site of pathology, helps to understand the molecular mechanism of diseases and providing a window of opportunity to identify potential biomarkers and therapeutic targets. 
     Tissue proteomics requires tissues to be stored by snap freezing. However, flash frozen tissues without embedding medium are difficult to section thereby making histopathology or immunohistochemistry evaluation difficult. Instead, tissues are embedded in optimal cutting temperature medium (OCT) or formalin-fixed and paraffin-embedded (FFPE) to retain its morphology (Turbett and Sellner, 1997). FFPE embedded tissues have been recently explored by various groups for proteomics analysis (Ralton and Murray, 2011; Vincenti and Murray, 2013). However, FFPE tissues during formalin fixation undergo extensive crosslinking between protein/DNA/RNA with methylene bridges creating inter and intra crosslinking of proteins (Turbett and Sellner, 1997; Magdeldin and Yamamoto, 2012). Some modifications of peptides in proteomics analysis of FFPE tissues are Metylol derivatives, Schiff bases and methylene bridges (Magdeldin and Yamamoto, 2012). Time span during FFPE process and storage can also lead to different levels of protein degradation and protein modifications. In contrast, OCT embedded tissues are instantly stored at freezers for histological studies; therefore, the protein contents are likely maintained and are representative of the tissue proteome. 
     However, the proteomic analysis of OCT-embedded tissues is difficult. OCT contains water soluble synthetic polymers and is widely used for embedding tissues for storage. OCT can compete with peptides for ionization during mass spectrometry analysis (Setou, 2010). OCT can also generate ion suppression in Matrix Assisted Laser Desorption and Ionization (MALDI) mass spectrometry and ionization competition in Electron spray ionization (ESI) mass spectrometry (Chaurand et al., 2004). In addition, OCT will create deleterious effect on the peptide chromatographic separation required for tissue proteomics. OCT has high affinity to reverse phase stationary medium commonly used in shotgun proteomics. OCT competes with peptides to bind to the column and prevails upon peptides for binding onto the C18 reverse phase column. OCT also decreases sensitivity of detection due to overlap with peptides during elution. For LC-MS/MS analysis of tissues, it is necessary to remove OCT from the sample. 
     In this study, a method is described using chemical immobilization of proteins for peptide extraction (CIPPE) from OCT-embedded tissues for tissue proteomic analysis. In this method, proteins are chemically immobilized onto solid support, which allows for sample cleaning and OCT removal by extensive washing before the peptides and modified peptides (glycopeptides) are released from the solid support using proteolysis. The method was applied to study the impact of OCT on tissue proteomics and glycoproteomics. 
     Materials and Methods 
     Materials: 
     Human fetuin, dithiotheritol (DTT), and iodoacetamide were purchased from Sigma Aldrich (St. Louis, Mo.). Rapigest was purchased from Waters (Milford, Mass.). Protein estimation BCA kit, sodium cyanoborohydride, and Aminolink coupling resin was purchased from (Thermo Fisher Scientific Inc., Rockford, Ill.). Sequencing grade trypsin was purchased from Promega (Madison, Wis.). iTRAQ 4-plex reagents were purchased from AB Sciex (Framingham, Mass.). PNGase F was obtained from New England Biolabs (Ipswich, Mass.). 
     Protein Extraction: 
     Mouse kidney tissue was collected from NIH01a mice and snap frozen in Dr. Kemp&#39;s laboratory of Fred Hutchinson Cancer Research Cancer (Tian et al., 2010; Tian et al., 2009). Mouse kidney tissue was cut into two pieces. One was embedded in OCT followed by storage at −80° C. The second piece was stored as fresh-frozen tissue. OCT embedded or frozen mouse kidney tissues was lysed in 500 μL of pH 10 tissue lysis buffer (100 mM sodium citrate and 50 mM sodium carbonate in 2% SDS) by vortexing for 2-3 min and sonicating for 4 min in an ice bath to homogenize the tissues. After the tissues were homogenized, BCA was used to estimate the protein concentration. 
     Chemical Immobilization of Proteins to Beads: 
     Proteins were immobilized on to amino-link beads using previously described protocol (Yang et al, submitted to MCP). Briefly, amino-link resin (800 μL) was loaded onto snap-cap spin-column, and centrifuged at 2000 g for 1 minute. Resin was washed with 800 μL of pH 10 buffer (sodium citrate 100 mM and sodium carbonate 50 mM buffer) followed by centrifugation. The washing step was repeated twice. The sample in pH 10 buffer 10 (1 mg/200 microliter sample to beads ratio) was loaded onto amino-link resin. Volume was adjusted to 850 μL using pH 10 buffer. 
     Sample-resin mixture was incubated at room temperature overnight on a mixer. The mixture was centrifuged at 2000 g to remove any unbound protein. Resin was rinsed by 1×PBS buffer (Sigma-Aldrich; pH 7.4; 450 μL) three times. 50 mM sodium cyanoborohydride in PBS (400 μL) was added to resin (spin-column capped during each incubation step). After a four hour incubation, supernatant was removed via centrifugation (2000 g) and 400 μL of 1 M Tris-HCl (pH 7.6) in the presence of 50 mM sodium cyanoborohydride was added to block any un-reacted aldehyde sites of resin. The blocking process was terminated after 1 hour. Then, the beads were washed with PBS twice, 1.5M of NaCl twice, and water three times. 
     Peptide Extraction by Proteolysis: 
     Proteins bound on the beads were treated with 10 mM DTT in 50 mM ammonium bicarbonate for 30 mins at 60° C. followed by a wash with 50 mM ammonium bicarbonate. Afterwards, the beads were treated for 1 hr with 15 mM iodoacetamide in 50 mM ammonium bicarbonate in dark. Finally, proteins were digested using 1:50 trypsin to protein ratio in presence of 0.1% rapigest with 50 mM ammonium bicarbonate. The proteins were digested at 37° C. overnight. The released peptides were collected from the supernatant of the beads and the following wash step of the beads with water. 
     Ammonium bicarbonate was evaporated using freeze drying before iTRAQ labeling. iTRAQ labeling was performed according to manufactures protocol. 
     In-Solution Digestion: 
     Human serum albumin (HSA) protein with and without OCT was incubated with 10 mM DTT at 60° C. for 1 hr, and alkylated with 30 min incubation 10 mM iodoacetamide in dark at room temp. Finally, the pH of the solution was adjusted to the 7.5 with 50 mM NH 4 HCO 3 . Protein was enzymatically digested with trypsin using 1:50 trypsin to protein ratio with incubation overnight at 37° C. 
     Mass Spectrometric Analysis of Peptides Using Direct infusion to TSQ Quantum: 
     A TSQ Quantum Ultra (Thermo scientific, Rockford, Ill.) with electrospray ionization source was used for analysis of peptides from HSA using direct infusion. Flow rate was set at 5 μL/min. Peptides were scanned from m/z 300 to 1000 at voltage of 3000 V and capillary temperature 180° C. was used for the spray. 
     N-Glycopeptide Enrichment: 
     N-linked glycopeptides were isolated from 90% of peptides of the iTRAQ labeled sample. Samples described above were treated using SPEG method (Tian et al, 2007). The enriched N-linked glycopeptides were concentrated by C18 columns and fractionated using basic reverse phase into 12 fractions and analyzed using LC-MS/MS. 
     High-pH RPLC Fractionation: 
     Fifty μg iTRAQ labeled peptides were submitted to high-pH RPLC fractionation with a 1200 Infinity LC (Agilent Technology, Santa Clara, Calif.) and a 4.6×100 mm BEH130-C-18 column (Waters, Milford, Mass.). Samples were adjusted to a basic pH using 1% ammonium hydroxide, and injected in 2 mls of solvent A 7 mM tri-ethyl ammonium bicarbonate (TEAB). Solvent B is 7 mM TEAB, 90% acetonitrile. 
     The separation gradient was set as following: 0% B for 18 min, 0 to 31% B in 42 min, 31 to 50% B in 10 min, 75 to 100% B in 15 mM, and 100% B for an additional 10 min. Ninety-six fractions were collected along with the LC separation and were concatenated into 24 fractions by combining fractions 1, 25, 49, 73, and so on. For glycoproteomic analysis, glycopeptides were concatenated into 12 fractions by combining every 13 th  fraction. The samples were dried in a Speed-Vac and stored at −80° C. until LC-MS/MS analysis. 
     LC-MS/MS Analysis: 
     Dionex Ultimate 3000 RELCnano system (Thermo Scientific, Rockford, Ill.) was used with a 75 μm×15 cm Acclaim PepMap100 separating column (Thermo Scientific, Rockford, Ill.). Peptides were separated using a flow rate of 300 nL/min with mobile phase A 0.1% formic acid in water and B consisting of 0.1% formic acid 95% acetonitrile. The gradient profile was set as follows: 4-35% B in 70 min, 35-95% B in 5 min. MS analysis was performed using an Orbitrap Velos Pro mass spectrometer (Thermo Scientific, Rockford, Ill.). The spray voltage was set at 2.2 kV. Orbitrap spectra were collected at a resolution of 60K followed by data-dependent HCD MS/MS (at a resolution of 7500, collision energy 45% and activation time 0.1 ms) of the ten most abundant ions. A dynamic exclusion time of 35 sec was used with a repeat count of 1. 
     Database Search: 
     Data generated using Orbitrap was searched using Proteome Discoverer 1.3 (Thermo Scientific, Rockford, Ill.) against IPI mouse database v3.30 with 56688 protein entries. Peptides were searched with two trypsin ends as protease, allowing only two missed cleavages. Search parameters used were 20 ppm precursor tolerance and 0.06 Da fragment ion tolerance, static modification of 4plex iTRAQ at N-terminus and Carbamidomethylation at Cysteine. Variable modification of oxidation at methionine and deamidation at aspargine and iTRAQ at lysine. Filters used for data analysis included peptide rank1, 2 peptides per protein, and 1% FDR threshold. For glycopeptides, NXS/T motif was used for further filtration of data. 
     Data Analysis of Removal of OCT from Human Serum Albumin (HAS): 
     Peaks were selected from ESI spectrum obtained from TSQ quantum with a threshold of 20% intensity of base peak intensity. Peaks were obtained from HSA protein digestion with OCT, without OCT, and with OCT followed by removal of OCT. Afterwards, they were aligned and compared. The comparison was performed between HSA, HSA with OCT, and HSA with OCT followed by OCT removal by CIPPE. 
     iTRAQ Data Analysis: 
     The Pearson&#39;s correlation coefficient of the peptide spectra obtained between replicated analyses of OCT embedded tissues (114, 115) using CIPPE was calculated to assess the reproducibility of the method to remove the OCT. Protein expressions in OCT embedded tissue (114, 115), and frozen tissue (116) were quantified and normalized by Proteome Discoverer 1.3. The log 2 ratios between replicates 114 and 115 were used as the “null” distribution, and the values for 5% cut-off (2.5th and 97.5th percentiles) of the histogram were selected as the thresholds for up- and down-expression thresholds. Similarly, the Pearson&#39;s correlation coefficient of the peptide spectra between the frozen tissue/OCT embedded tissues (116 and 114) was calculated to assess the impact of OCT embedding the tissue. The log 2 ratios between the frozen tissue/OCT embedded tissues (116 and 114) were compared with the up- and down-expression thresholds obtained in replicate analysis (“null” distribution). The same analysis protocol described above was applied to both the global proteomics data and the glycoproteomics data. 
     Results 
     Tissue proteomics is important for the identification of disease biomarkers, treatment targets and help in the understanding of the pathological characteristics of tissues. Currently, most of the tissue proteomic studies are performed on frozen tissues or FFPE embedded tissues. Due to the malicious effect of OCT to the mass spectrometer, only a handful of proteomics studies have been performed on OCT embedded tissues (Asomugha et al., 2010, Somiari et al., 2003; Nirmalan et al., 2011; Palmer-Toy et al., 2005; Scicchitano et al., 2009). OCT embedded tissues are studied using either two-dimensional gel electrophoresis (2D DIGE) technology or shot gun proteomics using LC-MS/MS. 2D DIGE could separate proteins from OCT; however, most of the LC-MS/MS studies of OCT embedded tissue had OCT contamination resulting in fewer protein identifications (Nirmalan et al., 2011; Palmer-Toy et al., 2005; Scicchitano et al., 2009). Recently, studies demonstrated that OCT embedded tissues could be used for glycoproteomic analysis using solid-phase extraction of glycopeptide (SPEG) (Tian et al., 2011). The glycopeptides were chemically immobilized to the solid support using oxidized glycan tags when the non-glycopeptides and OCT were removed from the immobilized peptides before the enzymatic release of N-glycopeptides. To analyze global proteome of tissues, a chemical immobilization of proteins for peptide extraction was employed based on the capture of proteins using beads containing amino groups ( FIG. 5 ). To remove OCT from the tissue sample, proteins were extracted from tissues and chemically immobilized onto the solid phase by reductive amination; however, inert OCT polymers from OCT-embedded did not get immobilized on the beads and was separated by washing the beads. Furthermore, the beads conjugated to proteins were reduced carbamidomethylated and proteolyzed to release the peptides for proteomics analysis ( FIG. 5 ). 
     To develop a procedure to remove the OCT, Human serum albumin (HSA) with and without OCT was used as a model protein. The tryptic peptides from HSA were directly analyzed by TSQ Quantum by direct infusion ESI.  FIG. 6A  shows the ESI spectrum of OCT contaminated HSA digested with trypsin demonstrating a regular bell shaped curve MS pattern with mass values of 44 Da, 22 Da and 14.6 Da apart. These clearly observed peaks correspond to different charge states of polyethylene glycol presented in OCT. OCT polymer overshadows the albumin peptides. In MS, OCT dominates the mass spectrum, indicating preferential ionization of OCT compared to albumin peptides. At 20% intensity of base peak, only 11 peptide peaks out of the 45 HSA peaks were detected in OCT contaminated HSA (10% OCT in volume/HSA weight). In contrast, HSA digest in OCT had 46 unique polymer peaks that suppressed the ionization of peptides and overshadowed these peptides in the mass spectrum. To remove OCT interferences from the sample, OCT contaminated HSA was first chemically immobilized onto beads using reductive amination, beads were then washed with various conditions, and the immobilized HSA was digested using trypsin. The released peptides were analyzed using ESI-MS ( FIG. 6B ). After washing beads with PBS, 1.5M NaCl and water, it was found that OCT peaks completely disappeared and HSA tryptic peptide peaks were visible in the mass spectrum. None of the 46 polymer peaks uniquely observed in OCT sample was observed after CIPPE. In this embodiment of the presently disclosed method, proteins were bound to solid phase and the inert OCT polymers were washed away, resulting in the complete removal of OCT form chemically immobilized proteins. The results showed that CIPPE removed OCT contaminants from protein sample, making high throughput proteomic analysis OCT-embedded tissues using mass spectrometry possible. However, it was observed that the fingerprint of tryptic peptides of albumin was different between CIPPE and in solution digest of HSA. Only 24 out of 45 HSA peptide peaks from non-OCT HSA were detected after OCT removal using CIPPE ( FIG. 6C ), which may have been due to OCT embedding or the sample process using CIPPE. 
     With the developed method to remove OCT contamination, the analysis of OCT embedded tissues was performed to study the impact of tissue embedding with OCT on proteomics and glycoproteomics. A complex biological tissue from mouse kidney was analyzed. Mouse kidney tissue was divided into two halves. One half was embedded in OCT and the other half was directly frozen. An OCT-embedded tissue (labeled with iTRAQ 114), a technical replicate of OCT-embedded tissue (labeled with iTRAQ115), and a frozen tissue (labeled with iTRAQ 116) were lysed and equal amount of proteins from the three tissues were used for quantitative proteomic profiling using chemical immobilization and iTRAQ methodology ( FIG. 7 ). Proteins from each sample were first bound to beads, followed by washing. Proteins were further reduced and alkylated on beads. Finally, proteins were released from beads using proteolysis, and the released peptides were iTRAQ labeled. Samples were split into two parts, 90% of sample was used for glycoproteomic analysis and 10% of sample was used for global proteomic analysis. In global proteomic analysis, basic reverse phase was used to generate twenty-four offline fractions, and each fraction was subjected to LC-MSMS analysis using Orbitrap Velos. In glycoproteomic analysis, the sample was subjected to glycopeptide enrichment using the SPEG method. Deglycosylated peptides were then analyzed using mass spectrometry ( FIG. 7 ). 
     From the global proteomic analysis of iTRAQ labeled tryptic peptides, 3857 proteins were identified on the basis of at least two peptides over thresholds score of 1% FDR. Quantification results are depicted in  FIG. 8 . Each dot represents a peptide spectra match. The replicates 114 and 115 showed little variance and little spread in the scatter indicating high quantitative reproducibility of the method ( FIG. 8A ). 95% of proteins showed ratio within the interval of 0.594 to 1.821 between 114 and 115. Equal percentage of the remaining proteins (i.e. 2.5%) fell either above 1.821 or below 0.594. The correlation between 114 and 115 channel was 0.92 for global proteomic analysis. From the analysis of replicate OCT-embedded tissues using CIPPE and MS/MS, it is estimated that proteins with changes beyond ratios of 0.59 and 1.83 are considered differentially expressed with 5% FDR. Using Orbitrap Velos, 468 unique glycosylated peptides were identified. Similarly, glycopeptides showed little variance ( FIG. 8B ) and 95% of glycoproteins showed a ratio within the interval of 0.21 to 2.44. Correlation between channels is 0.91 ( FIG. 8B ). These results indicated good analytical replications for global and glycoproteomics of OCT-embedded tissues using chemical immobilization. The results showed that accurate quantitation could be achieved on OCT embedded tissue using chemical immobilization, iTRAQ labeling, and tandem mass spectrometry. 
     The scatter plot of intensities of two channels 116 and 114 (frozen tissue/OCT embedded tissue) showed similar patterns as the technical replicates of OCT-embedded tissues ( FIGS. 8A and 9A ). The quantitative distribution are roughly symmetrical only with little spread from 1:1 line in the scatter plot, indicating high quantitative similarity between frozen and OCT-embedded tissue ( FIG. 9A ). A vast majority of the proteins belonged to 1:1 ratio irrespective of the intensity of iTRAQ channel and 86.36% of the proteins showed ratio within the interval of 0.59 to 1.83 (the same cut off from the replicate analysis). A percentage of 2.22 proteins showed a ratio above 1.821 while 11.41% of proteins displayed a ratio below 0.59. This percentage of down-regulated proteins indicated that there were apparently more peptides extracted from OCT embedded tissue compared to the frozen tissue. The correlation between 114 and 116 is 0.92 indicating good similarity from quantitation perspective for frozen and OCT-embedded tissues. Next, differential quantification of glycoproteome related to OCT embedded and frozen tissues was investigated.  FIG. 9B  shows the scatter plot of frozen tissues and OCT-embedded tissues for of the identified glycopeptides. The percentage of glycoproteins having a ratio between 0.21 and 2.44 (the same cut off from the replicate analysis) was 94.82%. Similar to quantitative analysis of global proteome, the quantitative distribution glycopeptide shows little variance indicating high quantitative similarity of glycoproteome between frozen and OCT embedded tissues ( FIG. 9B ). The correlation between 114 and 116 is 0.90, similar to replicate analysis of OCT-embedded tissues. To determine whether there were significant differences between OCT-embedded and frozen tissues, log 2 (116/114) in X axis and log 2 (115/114) in Y axis was plotted for global proteomics ( FIG. 9C ) and glycoproteomics ( FIG. 9D ). All proteins and glycoproteins are close to the origin. The results demonstrate that quantitative analysis of OCT embedded tissue is feasible. It has been shown that CIPPE is a method for quantitative analysis of protein expression and protein glycosylation in tissue proteomics from frozen and OCT-embedded tissues. Using this method, thousands of proteins from OCT-embedded tissues have been successfully identified. CIPPE has potential to be used for other PTM analysis like phosphorylation, ubiquitation and acetylation. 
     In addition to the removal of OCT from OCT-embedded tissues, this method could be used to extract proteins from tissues for tissue proteomics. Compared to the proteins from body fluids, the proteins from tissues are more difficult to extract in order to obtain a complete proteome due to the three-dimensional structures of tissues and solubility of certain tissue proteins. During the proteomic analysis of tissues, detergents such as sodium dodecyl sulfate (SDS), NP-40, or Triton X-100, are often used for protein extraction to solubilize the membrane proteins from tissues. However, detergents also distort mass spectrometric detection of peptides, similar to the observed spectra from OCT-contaminated HSA ( FIG. 6A ). In addition, these detergents, similar to OCT, bind to a reverse phase column, commonly used online with a mass spectrometer, further impairing the capability of tissue proteomics using LC-MS-MS/MS. CIPPE method is not only able to remove high concentration OCT, but also the detergents from the tissues samples introduced during the protein extraction for proteomics analysis. 
     In some cases, there is incomplete release of all tryptic peptides after the proteins are chemically immobilized onto the beads and peptides are released from beads using trypsin digestion. For protein identification and quantification, it is not necessary to recover all tryptic peptides. In the situations where all tryptic peptides are needed for the proteomic analysis, a cleavable linker to the solid phase could be used to capture and release all peptides. 
     This study shows that tissues embedded in OCT can be analyzed using shotgun proteomics. The CIPPE methodology described here was used to conduct global and glycoproteomics analyses of tissues embedded in OCT. When adopted, this protocol is highly efficient in the removal of contaminants Data indicated that OCT does not seem to impact the tissue proteome and glycoproteome. Therefore, CIPPE can be used for the analysis of OCT embedded tissue for proteomics and PTMs analysis like glycosylation, leading to the possibility of the discovery of potential biomarkers. 
       FIGS. 10-10B  show representative MALDI spectra of released tryptic global peptides released from casein immobilized to solid phase by reductive amination with a mass range of 500-4000 using an embodiment of the tube digestion method and the tip method. K.AVPYPQR (SEQ ID NO:355) is a peptide from beta casein. 
       FIGS. 11A-11B  show representative MALDI spectra of released tryptic peptides from casein immobilized to the solid phase in a tip with a mass range of 900-1700 using an embodiment of the tube digestion method and the tip method. R.FFVAPFPEVFGK (SEQ ID NO:357) and R.YLGYLEQLLR (SEQ ID NO:358) are peptides from alpha-S1-casein. 
     Example 3 
     High Throughput Analysis of N-Glycans Using Glycoprotein Immobilization for Glycan Extraction with Aldehyde Tips 
     Introduction 
     Aberrant glycosylation plays a critical role in many diseases where disease-associated glycans may be discovered for diagnosis and treatment. 
     To analyze N-glycans, a robust method for isolation of N-glyans using glycoprotein immobilization for glycan extraction (GIG) has been recently developed (Yang et al., 2013; Shah et al., 2013). Meanwhile, tip columns in combination with a robotic liquid handling system has shown its potential in high throughput sample processing for mass spectrometry analysis (Chen and Zhang, 2013). 
     To facilitate high throughput N-glycan analysis, a novel aldehyde tip was devised and tested for its performances on extracting N-glycans from human serum with a robotic liquid handling unit. 
     The incubation time for each of the major steps of N-glycan isolation was optimized, multiple parallel isolations of glycans were performed, the N-glycans extracted were analyzed by mass spectrometry and the reproducibility was assessed. 
     Methods 
     Preparation of Aldehyde Tip: 
     A round frit (2-mm-diameter and 1-mm-thick, pore size 15-45 microns) were first pushed into the pipette tip end (Disposable Automation Research Tips, Thermo Fisher Scientific, Waltham, Mass.). Two hundred microliters of aldehyde resin (50% slurry) was then loaded into each pipette tip. Liquids were blown out of the tip and a 5 mm round frit was pushed into the tip to secure the aldehyde resin between the two frits. Each tip was washed 5 times with 200 μL of water and conditioned 5 times with coupling buffer (100 mM sodium carbonate, pH=10) by aspirating and dispensing the solution. 
     Isolation of N-Glycans: 
     For protein immobilization, each tip was pipetted up and down in protein sample in sodium carbonate buffer (pH=10), followed by sodium cyanoborohydride containing PBS buffer (pH=7.4), and then Tris blocking buffer (pH=7.6). Sialic acid modification was performed by pipetting each tip up and down in p-toluidine solution (pH=4-6). For the washing step, each tip was pipetted up and down in 6 mL of 1% formic acid, 6 mL of 1M NaCl, 6 mL of 10% acetonitrile, and finally 6 mL of water. N-Glycan release occurred by pipetting each tip through 5 mM ammonium bicarbonate solution (pH=7.5) containing 2 μL PNGase F. The released N-glycans in the supernatant were collected and dried in vacuum. The extracted N-glycans were resuspended in HPLC grade water. 
     MALDI-MS Analysis: 
     N-glycans were analyzed using Axima MALDI Resonance mass spectrometer (Axima, Shimadzu, Columbia, Md.). Four microliters of dimethylamine (DMA) were mixed with 200 μL of 2,5-dihydrobenzoic acid (DHB) (100 μg/μL in 50% acetonitrile, 0.1 mM NaCl) as matrix-assisted laser desorption ionization (MALDI) matrix. Maltoheptaose (DP7) was spiked into each sample as a glycan standard at 25 mM. The laser power was set to 100 for two shots each in 100 locations per spot. The average MS spectra (200 profiles) were used for glycan assignment by comparing to the database of glycans previously analyzed by MALDI tandem mass spectrometry (MALDI-TOF-MS/MS). The assigned glycans were confirmed from human serum established in the literature. 
     Results 
       FIGS. 12A-12B  show an embodiment of a workflow scheme of N-glycan isolation. Proteins from samples were first immobilized onto beads/tip columns, sialic acid was then modified with p-toluidine, the beads/tips were subsequently washed extensively in 1% formic acid, 1M NaCl, 10% acetonitrile, and water, and the N-glycans were finally released with PNGase F. Photographs of an unpacked and packed aldehyde tip ( FIG. 13A ) and 96-well aldehyde tips loaded in a robotic liquid handling system for automated glycan extraction ( FIG. 13B ) are also shown. The reaction time for coupling and PNGase F release was optimized. Serum proteins were slowly pipetted through aldehyde tips for various amount of time and complete coupling was achieved after 30 min reaction ( FIG. 14A ). After extensive washing and sialic acid labeling, the N-glycans from serum proteins were released from the aldehyde tips with PNGase F for various times. N-glycan was still releasing after 2 hours ( FIG. 14B ). 
     MALDI-MS profiles of serum N-glycans isolated with the aldehyde tips were generated ( FIG. 15 ).  FIG. 16  shows representative MALDI profiles of three isolations of N-glycan from human serum. The glycans from the three isolations were quantified and the reproducibility of N-glycan isolation was assessed ( FIG. 17 ). 
     It was found that the application of aldehyde tips significantly reduced the processing time of N-glycan isolation and that aldehyde tips have great potential in achieving automation of N-glycan isolation for high throughput sample preparation when used in combination with liquid handling robotic systems. 
     Example 4 
     Solid Phase Labeling of Glycans and Proteins for Quantitative Glycopeptide Analysis Introduction 
     Glycosylation is one of the most abundant post-translational modifications on proteins. Sialic acids on glycoprotein are typically found at the terminal residue of glycans. Sialic acids play crucial role in cell surface interactions, protect cells from membrane proteolysis, help in cell adhesion, and determine half-life of glycoprotein in blood. The degree of sialylation has been demonstrated to be a consequence of diseases. 
     A strategy has been developed to label aspartic acid, glutamic acid and sialylated glycans with stable isotopic tags in a single process for quantitative MS analysis. A quantitative method of solid-phase sialic acid labeling is described ( FIG. 18 ). N-glycans were identified and quantified from SW1990 cells ( FIGS. 19A-19C ; SW1990 Cells with and without 1,3,4-O-Bu3ManNAc treatment). 87 N-glycans and 32 sialylated N-glycans were identified and 14 sialylated N-glycans were relatively quantified (Table 6). 
     Advantages of labeling include stabilization of the sialylated glycan and removal of the negative charge from N-glycans; the sample is first bound to the beads and hence the proteins after removal of N glycans can be analyzed using tryptic digestion; and along with sialic acid, aspartic acid and glutamic acid get modified and can be used for peptide/protein quantitation. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Sialylated N-glycans 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Sialic 
                 [M + 
                   
                   
               
               
                 Core + Na 
                 Fucose 
                 HexNAC 
                 Hexose 
                 Acid 
                 Na]+ 
                 H/L 
                 Stdev 
               
               
                   
               
               
                 Core + Na 
                 0 
                 2 
                 2 
                 1 
                 2043.89 
                 1.29 
                 0.44 
               
               
                 Core + Na 
                 1 
                 2 
                 2 
                 1 
                 2189.95 
                 1.09 
                 0.17 
               
               
                 Core + Na 
                 2 
                 2 
                 2 
                 1 
                 2336.01 
                 1.10 
                 0.13 
               
               
                 Core + Na 
                 1 
                 3 
                 2 
                 1 
                 2393.04 
                 1.95 
                 0.11 
               
               
                 Core + Na 
                 2 
                 3 
                 2 
                 1 
                 2539.10 
                 1.31 
                 0.10 
               
               
                 Core + Na 
                 1 
                 3 
                 3 
                 1 
                 2555.10 
                 1.31 
                 0.45 
               
               
                 Core + Na 
                 1 
                 2 
                 2 
                 2 
                 2570.21 
                 1.02 
                 0.13 
               
               
                 Core + Na 
                 3 
                 3 
                 2 
                 1 
                 2685.16 
                 1.00 
                 0.17 
               
               
                 Core + Na 
                 2 
                 3 
                 3 
                 1 
                 2701.15 
                 1.03 
                 0.23 
               
               
                 Core + Na 
                 1 
                 4 
                 3 
                 1 
                 2758.19 
                 1.66 
                 0.31 
               
               
                 Core + Na 
                 1 
                 3 
                 2 
                 2 
                 2773.31 
                 1.70 
                 0.18 
               
               
                 Core + Na 
                 2 
                 4 
                 3 
                 1 
                 2904.25 
                 2.04 
                 0.50 
               
               
                 Core + Na 
                 1 
                 3 
                 3 
                 2 
                 2935.36 
                 0.98 
                 0.21 
               
               
                 Core + Na 
                 3 
                 4 
                 3 
                 1 
                 3050.31 
                 1.39 
                 0.30 
               
               
                   
               
            
           
         
       
     
     Glycopeptide analysis was performed using basic reverse phase fractionation ( FIG. 21 ). Sample preparation including labeling was automated using liquid handling robotic systems ( FIG. 22 ). Results showed quantitation of AFNSTLPTHAQHEK (SEQ ID NO: 354) CD44 glycopeptide with triattenary sialylated peptide ( FIGS. 22-23 ). 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Results from Glycopeptide Analysis 
               
            
           
           
               
               
               
            
               
                   
                   
                 Number of  
               
               
                   
                 Sample 
                 samples 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 MSMS containing oxonium ions 
                 4069 
               
               
                   
                 MSMS containing sialylated  
                 547 
               
               
                   
                 oxonium ions 
                   
               
               
                   
                 Unique sialylated oxonium  
                 390 
               
               
                   
                 ions precursor 
                   
               
               
                   
                 Global proteins identified 
                 2681 
               
               
                   
                 Proteins down regulated due  
                 116 
               
               
                   
                 to ManNAc treatment 
                   
               
               
                   
                 Proteins upregulated due  
                 243 
               
               
                   
                 to ManNAc treatment 
               
               
                   
                   
               
            
           
         
       
     
     In summary, a comprehensive quantitative N-glycosylation analysis was performed using stable isotope labeling on both glycans and proteins (glycosite-containing peptide, glycans, and glycopeptides). 1,3,4-O-Bu3ManNAc resulted in an increase in sialylation at specific glycosites. 
     Example 5 
     Discussion 
     In some embodiments, the presently disclosed subject matter provides a pipette tip comprising a chemical moiety. In other embodiments, the presently disclosed subject matter provides a hydrazide bead packed pipette tip for rapid, reproducible, and automated N-linked glycopeptide isolations. Using bovine fetuin as a standard glycoprotein, the incubation time was determined for each major step of glycopeptide isolation. Using commercially available human serum, multiple parallel isolations of glycopeptides were performed using hydrazide tips with a liquid handling robotic system. It was determined that with the hydrazide tip, the processing time was significantly decreased from the original three to four day SPEG manual procedure to less than an eight hour automated process. In addition, it was demonstrated that the hydrazide tip could perform glycopeptide isolations in a reproducible manner. The hydrazide tip was compatible with liquid handling robotics and has great potential in the automation of glycopeptide isolations for high throughput sample preparation. 
     In addition, to facilitate high throughput N-glycan analysis, a novel aldehyde tip was devised and successfully extracted N-glycans from human serum with a robotic liquid handling unit. 
     Further, a quantitative method of solid-phase sialic acid labeling was described. p-toluidine was successfully used to modify the acid component of proteins and sialylated glycans with a reliable and robust method for quantitation of glycan and glycopeptide. 
     The presently disclosed methods have been shown herein to be useful for a variety of glycoproteins or polypeptides. 
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     Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.