Abstract:
Embodiments of the present invention provide devices and methods for detecting, identifying, distinguishing, and quantifying modification states of peptides using Surface Enhanced Raman Spectroscopy (SERS) and Raman spectroscopy. Additional embodiments provide strategies for chemically derivatizing post-translational modifications and detecting the chemically derivatized products using SERS. Applications of embodiments of the present invention include proteome wide modification profiling and analyses with applications in disease diagnosis, prognosis and drug efficacy studies, enzymatic activity profiling and assays.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     The present application is related to U.S. application Ser. No. 10/919,699, filed Aug. 16, 2004 and U.S. application Ser. No. 11/202,862, Filed Aug. 11, 2005. 
     
    
     FIELD OF THE INVENTION  
       [0002]     Embodiments of the present invention relate generally to the use of Raman spectroscopy for detecting, distinguishing, quantifying, and identifying modifications to and derivatives of amino acids, peptides, and proteins.  
       BACKGROUND OF THE INVENTION  
       [0003]     Post-translational modifications (PTMs) are believed to play an important role in the biological activity of proteins. Post-translational modifications are chemical processing events that cleave or add modifying groups to proteins for the purpose of modulating precise regulatory functions in a cell. Over 200 different types of PTMs have been described (see, for example, R. G. Krishna, F. Wold, in  PROTEINS: Analysis  &amp;  Design , Academic Press, San Diego, 121 (1998)) and PTMs such as acetylation (S. K. Kurdistani, S. Tavazoie, M. Grunstein,  Cell,  117, 721-733 (2004)), methylation (T. Kouzarides,  Curr. Opin. Genet. Dev.,  12, 198-209 (2002)), phosphorylation (P. Cohen,  Trends Biochem. Sci.  25, 596-601 (2000)), ubiquitination (P. Tyers, P. Jorgensen,  Curr. Opin. Genet. Dev.  10, 54-64 (2000)), and others play key roles in the regulation of gene expression, protein turnover, signaling cascades, intracellular trafficking, and cellular structure.  
         [0004]     In the past, mass spectrometry (MS) has been a favored approach for proteome-wide PTM profiling due to its sensitivity for measuring molecular weight changes in proteins and peptides. However, some modifications such as acetylation and trimethylation of lysine (both have nominal mass increases of 42 Da) and phosphorylation and sulfation of tyrosine (both have a nominal mass increases of 80 Da) require expensive, high-resolution mass spectrometers or require mass spectrometry analysis schemes that are not conducive to high-throughput analyses. Also, modifications such as phosphorylation, sulfation, and glycosylation are unstable during tandem mass spectrometry experiments making identification and positional information difficult to obtain. In few cases, quantification of protein expression and modifications using mass spectrometry has been performed using stable isotope labeling techniques. See, for example, S. P. Gygi et al.,  Nature Biotechnology,  17, 994 (1999) and X. Zhang, Q. K. Jin, S. A. Carr, Carr, R. S.,  Rapid Commun. Mass Spectrom.  16, 2325-32 (2002). Further, detection of phosphopeptides is hampered by their low ionization efficiency and ionization suppression leading to decreased sensitivity.  
         [0005]     Surface-enhanced Raman spectroscopy (SERS) is a sensitive method for chemical analysis. A Raman spectrum, similar to an infrared spectrum, consists of a wavelength distribution of bands corresponding to molecular vibrations specific to the sample being analyzed (the analyte). Raman spectroscopy probes vibrational modes of a molecule and the resulting spectrum, similar to an infrared spectrum, is fingerprint-like in nature. As compared to the fluorescence spectrum of a molecule which normally has a single peak exhibiting a half peak width of tens of nanometers to hundreds of nanometers, a Raman spectrum has multiple structure-related peaks with half peak widths as small as a few nanometers.  
         [0006]     To obtain a Raman spectrum, typically a beam from a light source, such as a laser, is focused on the sample generating inelastically scattered radiation which is optically collected and directed into a wavelength-dispersive spectrometer. Although Raman scattering is a relatively low probability event, SERS can be used to enhance signal intensity in the resulting vibrational spectrum. Enhancement techniques make it possible to obtain an approximately 10 6  to 10 14  fold Raman signal enhancement. Typically, a surface-enhanced Raman spectrum is obtained by adsorbing a target analyte onto a metal surface. The intensity of the resulting enhancement is dependent on many factors, including the morphology of the metal surface. Enhancements are achieved, in part, through interaction of the adsorbed analyte with an enhanced electromagnetic field produced at the surface of the metal.  
     
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0007]      FIG. 1  is a schematic diagram illustrating steps for protein profiling using SERS or Raman spectroscopy. Optionally, protein profiling may also include mass spectrometry.  
         [0008]      FIG. 2  maps post-translational modifications on the N-terminal tail of Histone H3 and indicates the biological significance of illustrated post-translational modifications.  
         [0009]      FIG. 3  shows the SERS spectrum of an unmodified peptide (P) (sequence:  9 KSTGGKAPR) with notations regarding the chemical bonding information that can be derived from the peaks (spectrum was taken at a peptide concentration of 9 ng/μl).  
         [0010]      FIG. 4  shows SERS spectra of unmodified and modified peptides (K9 peptide of the histone H3.3 of drosophila):  9 KSTGGKAPR(P),  9 K(trimethylated)STGGKAPR(P-9Me3), and  9 K(acetylated)STGGKAPR(P-9Ac). Spectra were taken a concentration of 9 ng/μl each. The spectra were arbitrarily offset along the y-axis for clarity.  
         [0011]      FIG. 5  shows the detection of trimethylated peptide, P-9Me3, at low concentrations. The spectra were arbitrarily offset along the y-axis for clarity. Arrows indicate strong spectral features that are present at all concentrations.  
         [0012]      FIG. 6A  provides SERS spectra of P-9Me2 ( 9 K(dimethylated)STGGKAPR) and P-9Me3 ( 9 K(trimethylated)STGGKAPR) peptide mixtures in which the concentration of P-9Me3 varied from 0% to 100%. The total concentration of the mixture was 70.0 ng/μL.  FIG. 6B  shows the quantification of post translational modification in mixtures of 9-trimethylated peptide P-9Me3,  9 K(trimethylated)STGGKAPR and 9-dimethylated peptide P-9Me2,  9 K(dimethylated)STGGKAPR. The Y-axis represents the ratio of intensities of peaks at 744 cm −1  and 1655 cm −1  from the SERS spectra of different concentration % mixtures. The X-axis represents the percent concentration of 9-trimethylated peptide P-9Me3 in the mixture.  
         [0013]      FIG. 7  diagrams a strategy for chemically derivatizing a phosphorylation post-translational modification.  
         [0014]      FIG. 8  provides a strategy for chemically derivatizing a phosphorylation post-translational modification.  
         [0015]      FIG. 9  compares SERS spectra obtained from an underivatized phosphopeptide, a Raman tag derivatized phosphopeptide, and the free Raman tag.  
         [0016]      FIGS. 10A and 10B  provide a method for chemical tagging of phosphopeptides and an exemplary Raman tag, respectively.  
         [0017]      FIG. 11  provides a method for the synthesis of diazo-compounds useful as Raman tags.  
         [0018]      FIG. 12  provides a method for affinity tagging phosphopeptides with a Raman label.  
         [0019]      FIG. 13  shows and exemplary scheme for the synthesis of an affinity ligand-Raman tag molecule.  
         [0020]      FIG. 14  shows an additional exemplary scheme for the synthesis of an affinity ligand-Raman tag molecule.  
         [0021]      FIG. 15  provides a chemical derivatization strategy for glycosylation post-translational modifications.  
         [0022]      FIG. 16  shows a scheme for affinity tagging glycosylation post-translational modifications.  
         [0023]      FIG. 17  provides some exemplary Raman tag molecules.  
         [0024]      FIGS. 18A and 18B  each diagram a use of SERS to detect peptide modifications. In  FIG. 18A , a substrate containing an array having a multiplexity of peptides at different locations is allowed to interact with a sample of biologic origin (containing, for example, enzymes or cell lysates), resulting posttranslational modifications are chemically derivatized, and SERS is performed before and after the enzymatic interaction and chemical derivatization. In  FIG. 18B , a peptide array is made from a digested set of proteins or biofluids deposited on a substrate, selected enzymes are reacted with the peptides of the array, resulting posttranslational modifications are chemically derivatized, and SERS is performed before and after the enzymatic interaction and chemical derivatization.  
         [0025]      FIG. 19  schematically describes a Raman spectrometer that can be used for SERS measurements.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0026]     A variety of modifications to the amino acid building blocks that make up a peptide or a protein are possible, such as for example, dimethylation, trimethylation, acetylation, phosphorylation, ubiquination, palmitoylation, glycosylation, lipidation, sulfation, and nitrosylation. (See also, for example, “Proteomic analysis of post-translational modifications,” Mann et al.,  Nature Biotechnology,  21:255 (2003)). Embodiments of the present invention provide the ability to detect modification(s) to the amino acids in a peptide at low concentrations, and also to distinguish, identify, and quantify them based on spectral signatures.  
         [0027]     In embodiments of the present invention, SERS and Raman analysis can be used alone or in conjunction with Mass spectrometry (for example, ESI (electrospray ionization) or MALDI (matrix-assisted laser desorption/ionization) mass spectrometry) to obtain modification or protein profiles of different biofluids after physical or affinity-based (e.g., antibody-based) separations for applications such as disease diagnosis and prognosis, and drug efficacy investigations. Referring now to  FIG. 1 , a flow chart is provided generally outlining a method for protein profiling according to an embodiment of the present invention. Typically, a sample obtained from a biologic source, such as for example, a bodily fluid or cell lysate solution, is a complex mixture of proteins and other molecules. The components of the mixture can be separated using known techniques for isolating proteinaceous fractions, the protein and peptide containing fractions, from biologic samples, such as for example, physical or affinity based separation techniques. The isolated proteinaceous fraction can then be digested into smaller peptides. Typical methods include enzymatic digestions using, for example, proteinase enzymes such as, Arg-C(N-acetyl-gamma-glutamyl-phosphate reductase), Asp-N, Glu-C, Lys-C, chromotrypsin, clostripain, trypsin, and thermolysin. The resulting digest of peptides can be further separated, for example, using HPLC (high pressure liquid chromatography). PTMs present on the digested peptides can be chemically derivatized to provide functional groups detectable by Raman spectroscopy. Raman spectroscopy or SERS is then performed on the resulting sample by, for example, mixing the digested sample with a SERS solution, such as for example, a colloidal silver solution, depositing and drying the digested sample onto a substrate and subsequently adding a SERS solution, such as a colloidal silver solution, depositing the sample onto a SERS-active substrate, or it can be performed in-line in a component of a microfluidic or nanofluidic system, such as by using a micro or nanomixer to mix the SERS solution with a the digested sample and subsequently performing Raman analysis on the sample. A silver colloidal solution can be mixed with digested sample eluants in a fluidic format, optionally, on a chip using microfluidics, and the detection can be performed inline as the eluants are flowing through the laser detection volume. In additional embodiments, some or all of these steps are performed using microfluidics.  
         [0028]     In general, the detection target or biologic sample can be found in any type of animal or plant, or unicellular organism. For example, biologic sample could be a mammalian cell such as an immune cell, a cancer cell, a cell bearing a blood group antigen such as A, B, D, or an HLA antigen, or virus-infected cell. Further, the detection target could be from a microorganism, for example, bacterium, algae, virus, or protozoan. The analyte may be a molecule found directly in a sample such as a body fluid from a host. The body fluid can be, for example, urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, and the like.  
         [0029]     Raman active surfaces of various forms can be used in embodiments of the present invention. For example, Raman active surfaces include, but are not limited to: a metallic surface, such as one or more layers of nanocrystalline and/or porous silicon coated with a metal or other conductive material; a particle, such as a metallic nanoparticle; an aggregate of particles, such as a metallic nanoparticle aggregate; a colloid of particles (with ionic compounds), such as a metallic nanoparticle colloid; or combinations thereof. Typical metals used for Raman enhancement include, silver, gold, platinum, copper, aluminum, or other conductive materials, although any metals capable of providing a SERS signal may be used. The particles or colloid surfaces can be of various shapes and sizes. In various embodiments of the invention, nanoparticles of between 1 nanometer (nm) and 2 micrometers (μm) in diameter may be used. In alternative embodiments of the invention, nanoparticles of 2 nm to 1 μm, 5 nm to 500 nm, 10 nm to 200 nm, 20 nm to 100 nm, 30 nm to 80 nm, 40 nm to 70 nm or 50 nm to 60 nm diameter may be used. In certain embodiments of the invention, nanoparticles with an average diameter of 10 to 50 nm, 50 to 100 nm or about 100 nm may be used.  
         [0030]      FIG. 2  maps PTMs on an exemplary protein fragment, the N-terminal tail of Histone H3. The biological significance of certain modifications of the N-terminal tail of Histone H3 in cellular functions such as, transcription, mitosis, and gene silencing, is indicated. SERS provides the ability to detect the presence of post-translational modifications of similar mass on peptides using SERS. For example, part of the N-terminal tail of histone H3 ( 9 KSTGGKAPR) (P) has lysines at the amino-acid positions 9 and 14 in this peptide that are frequently targeted for modifications such as acetylation and methylation and the serine and threonine at amino acid positions 10 and 11 respectively are targeted for phosphorylation. (See  FIG. 2  for a map of biologically significant modification sites.) These modifications are known to have major effects on the histone-histone as well as the histone-regulatory protein interactions (see for example, S. K. Kurdistani, S. Tavazoie, M. Grunstein,  Cell  117, 721-733 (2004); T. Kouzarides,  Curr. Opin. Genet. Dev.,  12, 198-209 (2002); B. D. Strahl, C. D. Allis,  Nature  403, 41-45 (2000); S. J. Nowak, V. G. Corces,  Trends in Genetics  20, 214-220 (2004); S. L. Berger,  Curr. Opin. Genet. Dev.,  12, 142-148 (2002); and Tamaru H. et al.,  Nat. Genet.  34, 75-79 (May 2003)).  
         [0031]      FIG. 3  shows the SERS spectrum of the unmodified peptide, the N-terminal tail of histone H3 ( 9 KSTGGKAPR). The peaks in the SERS spectrum can be assigned to different vibrational bands within the peptide (see, for example, S. Stewart, P. M. Fredericks,  Spectrochimica Acta Part A  55, 1615-1640 (1999); W. Herrebout, K. Clou, H. O. Desseyn, N. Blaton,  Spectrochimica Acta Part A  59, 47-59 (2003)). Particularly strong peaks can be observed at 919 cm −1  (C—COO − ), 1250 cm −1  (CH 2  wag), 1436 cm −1  (CH 2  scission) and 1655 cm −1  (Amide I).  
         [0032]      FIG. 4  shows a comparison of SERS spectra for the 9-trimethylated (P-9Me3) and 9-acetylated (P-9Ac) peptides to that of the unmodified peptide. The spectral signatures of the peptides differ based on the modification of a single amino acid. Peaks were observed in the SERS spectra of both the trimethylated and acetylated peptides that were absent from the spectrum of the unmodified peptide as indicated by the arrowheads in  FIG. 4 . As can be seen from  FIG. 4 , even though the mass difference between these modifications is only 0.03639 amu, they can be distinguished from one another. A very strong peak is observed at a wave-number of 744 cm −1  for the 9-trimethylated peptide, P-9Me3, due to the trimethyl modification (CH 3  terminal rocking) of the lysine. The high signal intensity of this peak is believed to be attributed to the strong interaction between the positively charged N-terminus and the trimethyl ammonium side chain with the negatively charged silver nanoparticles (the surface charge density (Zeta potential) for the silver colloidal particles were measured using a Zetasizer (Zetasizer Nano, Malvern) and found to be about 62±3 mV). In the case of the 9-acetylated peptide, P-9Ac, a strong peak is observed at a wave-number of 628 cm −1  that can be assigned to the side chain O═C—N bending resulting from the acetyl modification.  
         [0033]     Further, using SERS, zeptomoles (10 −21  moles) of the trimethylated modified peptide P-9Me3 can be detected. The ability to detect very low concentrations is useful because the stoichiometry of post-translational modifications can be very low.  FIG. 5  shows the spectra of the 9-trimethylated peptide P-9Me3 at different concentrations over three orders of magnitude ranging from 9 ng/μl to 9 pg/μl. Concentrations down to 9 pg/μl, which corresponds to about 10 fmol/μl, exhibit the same features (strong peaks at 744 cm −1  and 1436 cm −1 ) observed in spectra from higher concentrations of the 9-trimethylated peptide P-9Me3. A concentration of 9 pg/μl corresponds to about 10 zeptomoles of the 9-trimethylated peptide P-9Me3 in the collection volume of the laser beam (the collection volume of the laser illumination spot was estimated to be about 2.5 μm×2.5 μm×200 μm).  
         [0034]     SERS can also be used to quantify the concentrations of peptides having different modifications in a mixture. For example,  FIG. 6A  shows the SERS spectra of a mixture of 9-dimethylated peptide, P-9Me2 ( 9 K Me2 STGGKAPR) and 9-trimethylated peptide, P-9Me3 ( 9 K Me3 STGGKAPR). The unique peak at 744 cm −1  corresponding to the trimethylation modification from peptide P-9Me3 is visible in the spectra of the mixture. We performed quantification of trimethylation modification within the mixture using the SERS spectral information. SERS was performed on mixtures of different concentrations of 9-dimethylated and 9-trimethylated peptides, P-9Me2 and P-9Me3.  FIG. 6B  shows the graph of the ratio of the intensities at 744 cm −1  (corresponding to the trimethyl modification) and at 1655 cm −1  (corresponding to Amide I bending) plotted against the percent concentration of 9-trimethylated peptide P-9Me3. The linear trend for concentration versus peak intensity allows quantification of peptide concentrations in a sample by, for example, mapping peak intensity on a plot of known concentration versus peak intensity. This quantification ability allows, for example, enzymatic activity assays to be performed.  
         [0035]     It was found that factors, such as, for example, the addition sequence of the SERS cocktail and the incubation time on the SERS spectra of a modified peptide such as, the acetylated peptide (K(Acetylated)STGGKAPR), affected the intensity of the spectrum obtained. Additionally, the pH, ionic strength, and surface properties of the SERS substrate affect the spectrum obtained. In some embodiments of the present invention, the pH was controlled to have a delta less than about 0.5 pH and ionic strength was controlled, for example, about 20-300. In addition to the potential effects of pH changes on the spectroscopic and biochemical measurements, the effects of buffering capacity, which are dependent on the concentrations and the types of buffers, also play a role in determining the spectra obtained. For example, performing SERS in acidic condition (such as directly from an HPLC eluent of 0.1% TFA in ACN) increases the signal variations from chemical bonds that are closer to the N-terminal; while performing SERS using Ag particles coated with hydrophobic compounds (such as alkyl-thiol) magnifies the signal change from hydrophobic amino acid such as tyrosine. Also, the use of complexing agents such as divalent salts (Ca 2+ ) for masking or complexing with negative charges on a phosphorylation modification can help in bringing the biomolecule closer to the SERS substrate thereby increasing the ability to distinguish the modified peptide from an unmodified one.  
         [0036]     In embodiments of the present invention, post-translational modifications are modified by chemical derivatization and the modifications of the modifications are detected with SERS spectroscopy. A number of derivatization strategies are known for protein post-translational modifications. In some instances, chemical derivatizations can facilitate the detection of post-translational modifications by increasing the stability of the species to be detected and or presenting a species to be detected that provides enhanced signal characteristics in the detection strategy employed. In general, a molecule that can serve as a Raman tag is Raman active, preferably soluble in water, and positively charged. Advantageously, the applicability of embodiments of the present invention to the detection of chemical derivatizations of post-translational modifications is not limited to a particular type of derivatization employed.  
         [0037]     Referring now to  FIGS. 7 and 8 , a strategy for derivatizing a phosphorylation post-translational modification is provided. The strategy shown in  FIGS. 7 and 8  allow a phosphate group on a serine (Ser) or threonine (Thr) to be replaced by, for example, 2-methylaminoethanethiol, 2-dimethylaminoethanethiol, 2-trimethylaminoethane thiol, and 1H-benzotriazole-5-carboxylic acid (2-mercaptoethyl)-amide. (See, Oda, Y., Nagasu, T., Chait, B. T.,  Nat. Biotechnol.  19, 379-382 (2001); Meyer, H. E., Hoffmann-Posorske, E., Korte, H., Heilmeyer, L. M.,  FEBS Lett.,  204, 61-66 (1986); Simpson, D. L., Hranisavljevic, J., Davidson, E. A.,  Biochemistry,  11, 1849-1856 (1972); Byford, M. F.,  Biochem. J,  280 (Pt 1), 261-265 (1991); Adamczyk, M., Gebler, J. C., Wu, J.,  Rapid Commun. Mass Spectrom.,  15, 1481-1488 (2001); Goshe, M. B. et al.,  Anal. Chem.,  73, 2578-2586 (2001).) In  FIG. 8 , a Ser or Thr phosphorylation post-translational modification is replaced by 2-dimethylaminoethanethiol. (See, Steen, H. &amp; Mann, M.,  J. Am. Soc. Mass Spectrom.,  13, 996-1003 (2002).) The trimethylamino group, among other features of the resulting derivatized product, can be detected by SERS.  
         [0038]      FIG. 9  provides a comparison of spectra of an underivatized phosphopeptide, a Raman tag derivatized phosphopeptide, and the free Raman tag that was used to derivatize the phosphopeptide. In this example, the Raman tag was 2-dimethylaminoethanethiol. As can be seen from the spectra in  FIG. 9 , the derivatization of the phosphopeptide with the Raman tag provides a well-defined amplified signal indicating the presence of a phosphopeptide in the sample.  
         [0039]      FIG. 10A  provides a strategy for derivatizing a serine, threonine, or tyrosine (Tyr) phosphate posttranslational modification through reaction of a diazo compound with the proteinaceous phospho group. This strategy generally allows the phosphate group to be derivatized with a variety of Raman tags, for example, the compound shown in  FIG. 10B , 2-diazo-acetylamino-methyl-trimethylammonium chloride, and 4-diazomethyl-1-methyl-pyridinium chloride. The Raman tags can be chosen to provide strong detectable signals by SERS.  FIG. 11  provides a general synthesis scheme for making a diazo-Raman tag compound. In the example in  FIG. 11 , a trimethyl amine Raman tag having a diazo group for attachment to phosphates is created. (See also, Lansdell, T. A., Tepe, J. J.,  Tetrahedron Lett.,  45, 91-93 (2004).)  
         [0040]     Referring now to  FIG. 12 , a further method for modifying a phosphate to present a Raman-detectable tag is shown. In the method exemplified in  FIG. 12 , also known as affinity tagging, can be used in conjunction with any phosphorylated molecules. The metal for the ion shown in  FIG. 12 , M, can be any metal, including for example, Al, Ga, Fe, Ni, or Zn. In this example, the Raman tag is a Raman active structure that is preferably water soluble and positively charged.  FIG. 13  presents an additional method for synthesizing an affinity ligand Raman tag compound. (See, U.S. Pat. No. 6,623,655.) In the example shown in  FIG. 13 , an affinity ligand having a trimethyl amine Raman tag is created.  FIG. 14  demonstrates a further method for synthesizing an affinity ligand Raman tag compound. (See, U.S. Pat. No. 6,623,655.) In the example shown in  FIG. 14 , Rhodamine 6G, a Raman tag compound, is attached to an affinity ligand.  
         [0041]     In addition, phosphorylation PTMs can also be derivatized for example, by carbodiimide and imidazole with aminomethyl-trimethyl-ammonium chloride and aminoethyl-trimethyl-ammonium chloride.  
         [0042]     In further embodiments of the present invention, chemical derivatization strategies for glycosylation post-translational modifications, such as for example, O-linked N-acetylglucosamine, O-linked N-acetylgalactosamine are provided. (See, Wells, L. V. et al.,  Molecular and Cellular Proteomics,  1, 791-804 (2002).) In the example shown in  FIG. 15 , an O-linked N-acetylglucosamine (O-GlcNac) attached to a serine residue is derivatized with either dithiothreitol (DTT) or biotin pentylamine (BAP) tags. A similar strategy can be employed for chemical derivatization of glycosylation PTMs with different tags, such as for example, trimethylammonium, rhodamine dye, nitrobenzene, and diaminophenylazobenzene.  
         [0043]      FIG. 16  provides an additional method for attaching a Raman tag compound to a glycopeptide to facilitate the detection of a glycosylation PTM by Raman spectroscopy. In the method exemplified in  FIG. 16 , also known as affinity tagging, a boronic acid functionality bearing a Raman active species, is attached to a glycosylation PTM. The boronic acid functionality can have any structural format, and preferably the structure contains an aromatic ring. In further embodiments, the boronic acid affinity functionality can be replaced with a different sugar-binding structure, such as for example, lectins and concanavalin A.  FIG. 17  provides some exemplary Raman active tags.  
         [0044]     In addition, glycosylation PTMs can also be derivatized by periodate oxidation specific to the sugar moiety with a molecule containing a Raman active tag.  
         [0045]     In additional embodiments of the present invention enzymatic activity assays, such as, for example, phosphotase, kinase, acetylase, and deacetylase assays, are followed using chemical derivatization and SERS spectroscopy. For example,  FIG. 18  shows schematics illustrating two exemplary methods for enzymatic activity profiling. In  FIG. 18A , an array containing known peptides is synthesized using, for example, photolithography or spotting techniques, and is used as the substrate for testing the activity, such as for example detection or quantification of the activity of different types of enzymes, such as, for example, kinases, or phosphatases, or cell lysates or other samples of biologic origin. In a second example shown in  FIG. 18B , the array is comprised of unknown peptides obtained from the digestion of proteins or biofluids. The array can be made, for example, by spotting the sample containing the digested material onto a substrate, using for example, a commercially available array spotter. The substrate, for example, is a silver or gold surface and the peptides are attached through metal-thiol linkages. Additionally, the substrate could be a porous silicon surface having a gold or silver layer. After the enzyme or lysate is allowed to interact with surface-attached peptides, PTMs are chemically derivatized to provide labels that can be detected by Raman spectroscopy. SERS is performed before and after the enzymatic or lysate activity on the substrate peptide array and subsequent chemical derivatization, to determine the activity of particular enzymes on particular substrate peptides or lysates on particular peptides. In the case of peptides attached to a gold or silver surface, SERS is performed, for example, by depositing SERS active metal particles on the surface. The SERS particles can then be removed, for example by washing them from the surface, and the enzyme assay performed. SERS is then performed again by depositing SERS active metal particles once again on the substrate surface. In the case of the metal-coated porous silicon substrate, the substrate can act as an enhancement vehicle or the SERS active metal particles can be deposited on the surface. The activity of particular enzymes is determined and profiles are generated from different biofluids.  
         [0046]     Array compositions may include at least a surface with a plurality of discrete substrate sites. The size of the array will depend on the end use of the array. Arrays containing from about 2 to many millions of different discrete substrate sites can be made. Generally, the array will comprise from two to as many as a billion or more such sites, depending on the size of the surface. Thus, very high density, high density, moderate density, low density or very low density arrays can be made. Some ranges for very high-density arrays are from about 10,000,000 to about 2,000,000,000 sites per array. High-density arrays range from about 100,000 to about 10,000,000 sites. Moderate density arrays range from about 10,000 to about 50,000 sites. Low-density arrays are generally less than 10,000 sites. Very low-density arrays are less than 1,000 sites.  
         [0047]     The sites comprise a pattern or a regular design or configuration, or can be randomly distributed. A regular pattern of sites can be used such that the sites can be addressed in an X-Y coordinate plane. The surface of the substrate can be modified to allow attachment of analytes at individual sites. Thus, the surface of the substrate can be modified such that discrete sites are formed. In one embodiment, the surface of the substrate can be modified to contain wells or depressions in the surface of the substrate. This can be done using a variety of known techniques, including, but not limited to, photolithography, stamping techniques, molding techniques and microetching techniques. As will be appreciated by those in the art, the technique used will depend on the composition and shape of the substrate.  
         [0048]     A non-limiting example of a Raman detection unit is disclosed in U.S. Pat. No. 6,002,471. An excitation beam is generated by either a frequency doubled Nd:YAG laser at 532 nm wavelength or a frequency doubled Ti:sapphire laser at 365 nm wavelength. Pulsed laser beams or continuous laser beams can be used. The excitation beam passes through confocal optics and a microscope objective, and is focused onto the flow path and/or the flow-through cell. The Raman emission light is collected by the microscope objective and the confocal optics and is coupled to a monochromator for spectral dissociation. The confocal optics includes a combination of dichroic filters, barrier filters, confocal pinholes, lenses, and mirrors for reducing the background signal. Standard full field optics can be used as well as confocal optics. The Raman emission signal is detected by a Raman detector that includes an avalanche photodiode interfaced with a computer for counting and digitization of the signal.  
         [0049]     Another example of a Raman detection unit is disclosed in U.S. Pat. No. 5,306,403, including a Spex Model 1403 double-grating spectrophotometer with a gallium-arsenide photomultiplier tube (RCA Model C31034 or Burle Industries Model C3103402) operated in the single-photon counting mode. The excitation source includes a 514.5 nm line argon-ion laser from SpectraPhysics, Model 166, and a 647.1 nm line of a krypton-ion laser (Innova 70, Coherent).  
         [0050]     Alternative excitation sources include a nitrogen laser (Laser Science Inc.) at 337 nm and a helium-cadmium laser (Liconox) at 325 nm (U.S. Pat. No. 6,174,677), a light emitting diode, an Nd:YLF laser, and/or various ions lasers and/or dye lasers. The excitation beam can be spectrally purified with a bandpass filter (Corion) and can be focused on the flow path and/or flow-through cell using a 6× objective lens (Newport, Model L6X). The objective lens can be used to both excite the Raman-active probe constructs and to collect the Raman signal, by using a holographic beam splitter (Kaiser Optical Systems, Inc., Model HB 647-26N18) to produce a right-angle geometry for the excitation beam and the emitted Raman signal. A holographic notch filter (Kaiser Optical Systems, Inc.) can be used to reduce Rayleigh scattered radiation. Alternative Raman detectors include an ISA HR-320 spectrograph equipped with a red-enhanced intensified charge-coupled device (RE-ICCD) detection system (Princeton Instruments). Other types of detectors can be used, such as Fourier-transform spectrographs (based on Michaelson interferometers), charged injection devices, photodiode arrays, InGaAs detectors, electron-multiplied CCD, intensified CCD and/or phototransistor arrays.  
         [0051]     In certain aspects of the invention, a system for detecting the target complex of the present invention includes an information processing system. An exemplary information processing system may incorporate a computer that includes a bus for communicating information and a processor for processing information. The information processing and control system may further comprise any peripheral devices known in the art, such as memory, display, keyboard and/or other devices.  
         [0052]     While certain methods of the present invention can be performed under the control of a programmed processor, in alternative embodiments of the invention, the methods can be fully or partially implemented by any programmable or hardcoded logic, such as Field Programmable Gate Arrays (FPGAs), TTL logic, or Application Specific Integrated Circuits (ASICs). Additionally, the disclosed methods can be performed by any combination of programmed general purpose computer components and/or custom hardware components.  
         [0053]     Following the data gathering operation, the data is typically reported to a data analysis operation. To facilitate the analysis operation, the data obtained by the detection unit will typically be analyzed using a digital computer such as that described above. Typically, the computer will be appropriately programmed for receipt and storage of the data from the detection unit as well as for analysis and reporting of the data gathered.  
         [0054]     In certain embodiments of the invention, custom designed software packages can be used to analyze the data obtained from the detection unit. In alternative embodiments of the invention, data analysis can be performed using an information processing system and publicly available software packages.  
       EXAMPLE 1  
       [0055]     SERS experiments were performed as follows.  
         [0000]     Colloidal Silver Preparation  
         [0056]     Colloidal silver suspension was prepared by citrate reduction of silver nitrate as described in Lee and Meisel (P. C. Lee, D. J. Meisel,  Phys. Chem.  86, 3391 (1982)). The suspension had a final silver concentration of 1.00 mM. The surface charge density (Zeta potential) for the colloidal silver particles, after diluting 20 times with deionized (DI) water, was found to be 62±3 mV using a Zetasizer (Zetasizer Nano, Malvern).  
         [0000]     Peptide Synthesis  
         [0057]     Peptides with and without modifications were synthesized using Solid Phase Peptide Synthesis (SPPS) methods with standard Fmoc/t-buty/trityl protection chemistries to build up a full-length peptide chain. The starting amino acid was bound to a solid resin support (usually polystyrene) and its alpha amino group was chemically “blocked” with the Fmoc protecting group. Reactive side-chains were blocked with either t-Butyl or Trityl groups. The alpha-amino Fmoc protecting group was removed and an incoming amino acid (which was chemically activated on its carboxyl terminus to form an active ester) condensed to form a peptide bond. The process was repeated until the full-length product was obtained. The resin-bound peptide was then treated with trifluoroacetic acid (TFA) to remove the side-chain protecting groups and cleave the peptide from the polystyrene resin. Peptides were then precipitated out of solution with MTBE (methyl tertiary butyl ether) and lyophilized to dryness. For synthesis of modified peptides, trimethylated amino acid analogs were bought from Bachem in Switzerland, phospho-amino acids and acetyl-lysine were purchased from Nova Biochem in San Diego, Calif. Reverse-phase HPLC was utilized to purify and separate the target peptide from a crude mixture. MALDI-TOF mass spectrometry was used to determine the peptide&#39;s mass and compare with the expected peptide mass to confirm fidelity of the synthesis and purity of the product.  
         [0000]     SERS Measurements  
         [0058]     Peptides lyophilized after synthesis were resuspended in DI water at a concentration of 1 μg/μl and diluted to various sample concentrations. The stock solution of the synthesized colloidal silver, with a final silver concentration of 1.00 mM, was diluted 1 part to 2 parts in volume of DI water. Typically, 10 μl of the peptide solution was incubated with 80 μl of the diluted silver solution for 15 min. 20 μl of 0.5 M LiCl solution was added after the incubation and the solution was mixed thoroughly and dropped onto an aluminum tray for immediate SERS measurements. The laser was focused inside the sample droplet and 50-100 spectra were collected for each peptide sample. Typical collection time of each spectrum was 1 sec. Background was subtracted from the spectra by fitting an arbitrary linear baseline. Intensities of the peaks were calculated directly from the raw spectra by calculating the distance between the apex of the peak area and the midpoint of the base points of the peak area.  
         [0059]      FIG. 19  shows a schematic of a Raman spectrometer setup that was used for the SERS measurements. The system consisted of a titanium:sapphire laser  10  (Mira by Coherent, Santa Clara, Calif.) operating at 785 nm with power levels of about 750 mW, and a 20× microscope objective  20  (Nikon LU series) to focus the laser spot onto the sample plane. The peptide sample  30  was placed on an aluminum substrate  40 . The excitation beam  50  was filtered by a dielectric filter  60  (Chroma Technology Corp., Brattleboro, Vt.), to suppress spontaneous emission from the laser and transmitted through a dichroic mirror  60  (Chroma Technology Corp., Brattleboro, Vt.). The Raman scattered light from the sample  70  was collected by the same microscope objective  20 , and was reflected off the dichroic mirror  60  toward a notch filter or bandpass filter  80  (Kaiser Optical Systems, Ann Arbor, Mich.). The notch filter blocked the laser beam and transmitted Raman scattered light. The Raman-scattered light was imaged onto the slit of a spectrophotometer  90  (Acton Research Corp., Acton, Mass.) connected to a thermo-electrically cooled charge-coupled device (CCD) detector (Princeton Instruments, Princeton, N.J.) (not shown). The CCD camera was connected to a PC (not shown), and the collected spectrum was transported to the PC for visual display and computational analysis.  
       EXAMPLE 2  
       [0060]     The detection of post-translational modifications from biological samples was performed as follows.  
         [0000]     Enzymatic Digestion of Histone H3  
         [0061]     Lyophilized Histone H3 (obtained from Roche Applied Science, Inc.) was reconstituted in DI water to a concentration of 5 μg/μl. 5 μl of the reconstituted Histone H3 was digested with 250 ng of Endoproteinase Arg-C (enzyme substrate ration of 1:100 in a total volume of 50 μl of 50 mM ammonium bicarbonate buffer. Digestions were carried out at 37° C. for 16 hours. Digestion was halted by adding trifluoroacetic acid (TFA) to the digestion mixture at a final concentration of 0.5%.  
         [0000]     HPLC Separation of Digested Histone H3  
         [0062]     HPLC separation of the peptides from the digested Histone H3 was performed using an Alltech C18 column (150 mm×4.6 mm) using a two-step gradient. The gradients increased from 2 to 65% B over 63 min., stayed at 65% B for 7 min., and then increased from 65 to 85% B over 5 min. Solution A was 0.1% TFA in water and Solution B was 0.065% TFA in acetonitrile. Detection wavelength was 210 nm. Flow rate was 500 μl/min. Fractions were collected using an automated fraction collector every 10 s and combined according to peak positions and elution time. The combined fractions were then lyophilized to get rid of the mobile phase and then resuspended in 5 μl DI water for subsequent SERS and MALDI-TOF experiments.  
         [0000]     SERS Measurements  
         [0063]     Peptides lyophilized after synthesis and HPLC fraction collection were resuspended in DI water and diluted to various sample concentrations. The stock solution of the synthesized colloidal silver, with a final silver concentration of 1.00 mM, was diluted 1 part to 2 parts in volume of DI water. Typically, 10 μl of the peptide solution was incubated with 80 μl of the diluted silver solution at room temperature for 15 min. 20 μl of 0.5 M LiCl solution was added after the incubation and the solution was mixed thoroughly and dropped onto an aluminum plate for immediate SERS measurements. The laser was focused inside the sample droplet and 50-100 spectra were collected for each peptide sample. Typical collection time of each spectrum was 1 sec. Background from the spectra was subtracted by fitting an arbitrary linear baseline (shown in  FIG. 14A ). Intensities of the peaks were calculated directly from the raw spectra by calculating the distance between the apex of the peak area and the midpoint of the base points of the peak area ( FIG. 14B ).  
         [0064]     SERS measurements were performed as on a Raman spectrometer described in Example 1 and  FIG. 17 .  
         [0000]     Maldi-TOF Measurements  
         [0065]     Samples were spotted onto a target and MALDI data were collected on a Voyager DE-Pro mass spectrometer (Applied Biosystems) operated in reflection mode and calibrated externally.  
       EXAMPLE 3  
       [0000]     Chemical Derivatization of a Phospho Peptide  
         [0066]     A sample of a phosphorylated peptide (KRpTIRR, DLDVPIPGRFDRRVpSVAAE, or IGEGpTYGVVYK) was re-suspended in a 4:3:1 water:DMSO:ethanol solution to a total volume of 10 μL. 4.6 μL f a saturated barium hydroxide solution and 1 μL of a 500 mM sodium hydroxide solution were added. The reaction was allowed to incubate at about 37° C. (or at room temperature) for two hours and the solution was vortexed every 20-30 minutes. The reaction was cooled to room temperature. Then, 10 μL of a freshly prepared 1 M cysteamine-HCl or 1 M trimethyl cysteamine-chloride in deionized water solution was added and incubation was allowed to continue at room temperature for 3-6 hours. The reaction was monitored for completion and when the endpoint was reached, the reaction solution was diluted to 100 μL with 0.5% trifluoroacetic acid (TFA). The reaction was then purified on a micro-column and the resulting products were analyzed by MALDI. SERS was performed as above.  
         [0000]     Chemical Derivatization of a Phosphopeptide  
         [0067]     To 100 μL of a peptide stock solution containing 1.0 μg/μL DLDVPIPGRFDRRVpSVAAE in water was added 100 μL of a 180 mM (saturated, filtered, fresh, under nitrogen) Ba(OH) 2  solution. The reaction was incubated at room temperature until completion. Reaction progress was monitored by LC/MS. A C18 resin was used to desalt the resulting product. A Michael addition was performed by adding 0.2 M freshly prepared N,N,N-trimethyl cysteamine-chloride (thiol choline) solution in deionized water under nitrogen to the completed beta-elimination mixture. The resulting volume ratio was peptide:Ba(OH) 2 :thiol choline equal to 1:1:1, and the final pH 8.5. The reaction was allowed to incubate at room temperature until completion. Reaction progress was monitored by LC/MS. A C18 resin was used to desalt the resulting product. The derivatized peptide was purified by HPLC using the following protocol. Column: Agilent Zorbax C18 150 mm×0.5 mm, Flow rate 20 μl/min, Solvent A (aqueous): 0.1% formic acid (Fluka 94318) in HPLC grade water (from J.T. Baker cat# 4218), Solvent B (organic): 0.1% formic acid in acetonitrile (from Burdick and Jackson, cat #015-1), On-line detection: UV absorption at 214 nm and 280 nm simultaneously, Sample preparation: dissolve or dilute peptide samples with solvent A. Normal injection load of peptides for this capillary column: 10 μg. SERS was performed as described above.