# Patent

## Title
Detection and quantitation of full-length thioredoxin (TRX) and truncated thioredoxin (TRX 80) in complex samples

## Abstract

The present invention relates, e.g., to a method for detecting a full-length protein and a truncated form (e.g., a naturally occurring cleavage product) thereof, in a sample, comprising

    
    
        optionally denaturing and reducing proteins in the sample,
        cleaving the proteins into smaller peptides, and
        detecting a unique peptide identifier for the full-length protein and/or a unique peptide identifier for the truncated protein, in the sample.
    
    


In one embodiment of the invention, the full-length protein is thioredoxin (TRX), and the truncated form thereof is its biologically active, C-terminal truncated, 10 kDa cleavage product, TRX 80.

## Background
This application claims the benefit of the filing date of U.S. provisional application 61/264,344, filed Nov. 25, 2009, which is incorporated by reference herein in its entirety.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 17, 2011, is named 22429323.txt and is 9,987 bytes in size.
FIELD OF THE INVENTION
The present invention relates, e.g., to a method for detecting two forms of a protein in a sample, using unique peptide identifiers for the two forms of the protein.
BACKGROUND INFORMATION
Thioredoxin (TRX) is a 12 kDa redox cycling enzyme that is ubiquitously expressed in all cell types and is part of a key system for maintaining a reducing intracellular environment, working in a coordinate manner with thioredoxin reductase and NADPH (collectively referred to as the TRX system). TRX plays a role in regulating many different signaling processes, including cell cycle signaling, apoptosis, and glucose metabolism (Holmgren et al. (2010) Biochem Biophys Res Commun. 396, 120-124). Oxidative stress leads to TRX up-regulation, which then can be secreted by a yet-undefined leaderless pathway. TRX 80, a 10 KDa cleavage product of secreted TRX thought to be composed of the 80 N-terminal amino acids, is produced mainly from cleavage of secreted TRX by activated monocytes/macrophages (Silberstein et al. (1987) J Immunol 138, 3042-3050; Silberstein et al. (1989) J Immunol 143, 979-983). TRX 80 has monocyte chemoattractant activity and induces differentiation and activation of monocytes to a highly inflammatory phenotype that produces TNFα, IL-1β, IL-6 and IL-8, termed the TRX 80 activated monocyte (TAM) (Pekkari et al. (2005) Blood 105, 1598-1605). These cells also upregulate the costimulatory molecule CD86, and when cocultured with T cells produce IL-12 and aid in stimulating interferon-γ production (Pekkari et al. (2005) (supra); Pekkari et al. (2001) Blood 97, 3184-3190), suggesting that TAMs facilitate the generation of TH1 (proinflammatory) lymphocytes.
Increased plasma TRX levels have been demonstrated in many disease conditions, including heart failure, cardiomyopathy, cancer, asthma, and rheumatoid arthritis, among others, and are thought to be highly indicative of oxidative stress (Yoshida et al. (1999) J Immunol 163, 351-358; Kishimoto et al. (2001) Jpn Circ J 65, 491-494; Jikimoto et al. (2002) Mol Immunol 38, 765-772; Yamada et al. (2003) Immunol Lett 86, 199-205; Miwa et al. (2005) Circ J 69, 291-294; Grogan et al. (2003) Hum Pathol 31, 475-481). TRX has been tested as an oxidative stress marker in a study examining of the administration of A-type natriuretic peptide to reduce oxidative stress in heart failure patients (Shono et al. (2007) Circ J 71, 1040-1046).
Examination of TRX 80 levels in human disease or in model systems has been somewhat limited to date. The cleavage of TRX to TRX 80 may have profound influence on local sites of oxidative stress. TRX 80 levels were shown to be highly variable in plasma of a small number of presumed-healthy donors (Pekkari et al. (2000) J Biol Chem 275, 37474-37480). Additionally, production of TRX 80 by synoviocytes cultures from patients with rheumatoid arthritis (RA) has been demonstrated (Lemarechal et al. (2007) Clin Sci (Lond) 113, 149-155). Elevated levels of secreted TRX previously have been associated with RA (Jikimoto et al. (2002) (supra)), and in the aforementioned study by Lemarechal and colleagues, TRX 80 was produced at basal levels by cultured synovial cells obtained from RA patients, but not from osteoarthritis patients, with production increased by IL-1β and/or TNF-α stimulation. This suggests a role for TRX 80 production in the infiltration and proliferation of immune cells that are hallmarks of RA.
Several experimental approaches have been used to identify TRX and/or TRX 80 in biological samples, each with its own strengths and limitations. Due to their different functions, it is important to distinguish TRX from TRX 80. However, as TRX 80 is derived from the same amino acid sequence as TRX and they are relatively close in size (12 vs. 10 kDa), it can be difficult to clearly resolve them in a standard immunoblotting assay. Even antibodies developed to specifically recognize TRX 80 in its native nondenatured form may recognize both TRX 80 and TRX in western blot (Sahaf et al. (1997) Exp Cell Res 236, 181-192).
ELISA is frequently used to detect TRX. While potentially informative, most ELISAs may crossreact to TRX 80. One group has designed an ELISA that specifically detects the truncated form TRX 80. However, this assay still shows a very small level of cross reactivity for the full length protein (Pekkari et al. (2000) (supra)), which can be problematic if TRX 80 needs to be accurately monitored and is present at much lower levels than TRX.
Other TRX properties may contribute to inaccurate quantitation by immunologic detection. In plasma, TRX 80 may be present in protein dimers (Pekkari et al. (2000) (supra)). TRX also can be bound to other proteins such as albumin, hindering its detection in nondenaturing sample processing methods.
To clearly define a role for TRX in disease, and to use the detection of TRX and/or TRX 80 to diagnose a disease, monitor its progress, or the like, it is important to be able to simultaneously monitor the presence of both TRX forms in a quantitative capacity in complex samples, including plasma and tissue homogenates. There is a need for an assay that can detect the two forms of TRX, unambiguously and quantitatively, e.g. within the same sample.



DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1F shows a reference chart for TRX and TRX 80. The sequences of the unique peptides produced by CNBr digestion are provided for both TRX and TRX 80, as well as the resulting MS peaks obtained by MALDI and their MS/MS spectra with labeled b and y ions (representative spectra shown). The sequence of full length thioredoxin is SEQ ID NO:3. The sequence of TRX 80 is SEQ ID NO:4. The sequences of the shared CNBr peptides are SEQ ID NO:5 (upper) and SEQ ID NO:6 (lower). The sequence of the unique peptide for full length TRX is SEQ ID NO:1. The sequence of the unique peptide for TRX 80 is SEQ ID NO:2. Synthetic peptide standards containing heavy labeled lysine (SEQ ID NOS:34-35, respectively, in order of appearance)(*K, 13C6 15N2) were later produced for these same sequences to be used for quantitation in an MRM assay, as is discussed elsewhere herein.
FIGS. 2A-2F shows an experiment in which CNBr digests of recombinant TRX and TRX 80 spiked into 100 μg of CNBr-digested whole human plasma, in consecutive 10 fold dilutions, were fractionated by HPLC-SCX. The identifying peptide for TRX 80 (914 kDa) consistently eluted at 9 minutes on a 35 minute linear gradient (0-350 mM KCl) and was detected at levels down to 10 ng from the complex plasma mixture, while that for TRX (3484 kDa) eluted at 34 minutes and was detected down to 1 ng (representative spectra shown, experiment performed in duplicate).
FIGS. 3A-3E shows LC-MRM quantitation curves in plasma. MRM transitions were monitored for dilutions of heavy standard peptides in CNBR-digested plasma (1-10,000 fmole peptide in 2 μL of plasma protein). XICs are shown at the lower limit of quantitation, defined by CV<20% and accuracy between 80-120%). Curves were produced in triplicate using high-flow LC followed by MRM on the QTRAP 5500 system.
FIGS. 4A-4C shows LC-MRM quantitation curves in CSF. MRM transitions were monitored for dilutions of heavy standard peptides in CNBR-digested CSF. XICs are shown at the lower limit of quantitation (LLOQ, defined by CV<20% and accuracy between 80-120%). Curves were produced in triplicate using high-flow LC followed by MRM on the QTRAP 5500 system.
FIG. 5 shows CSF TRX levels measured by MRM. TRX was measured in CSF samples from uninfected, SIV-infected, and SIV-infected minocycline treated macaques that went on to develop either mild/none or moderate/severe encephalitis. Group median values are displayed for each group (bar) Animals that developed moderate or severe encephalitis had significantly elevated levels of CSF TRX (p=0.05; Mann-Whitney).
FIG. 6 shows protein digestion with CNBr. Western blot shows the detection of intact TRX and TRX 80 with an anti-thioredoxin antibody (individually, and spiked into plasma), and the loss of immunoreactivity with CNBr digestion, supporting effective cleavage both in buffer and in the matrix of plasma (top). Parallel total protein staining (SYPRO Ruby) shows that recombinant TRX and TRX 80 are effectively digested by CNBr (bottom, representative gel). Expected fragment masses with CNBr digestion are 3484, 3951, and 4390 Da for TRX, and 914, 3951, 4390 Da for TRX 80 (with cysteine alkylation, and homoserine lactone [HSL] formation due to CNBr digestion). A 914 kDa band could not be resolved, although it was later detected by MS. Digestion of the spiked plasma also is illustrated, showing a general loss of high molecular weight protein species and an increase in low molecular weight peptides.
FIGS. 7A-7F shows the limits of detection of unique peptides from CNBr-digested TRX and TRX 80 by MALDI-TOF. Both peptides were clearly detected from digests of 50 ng of recombinant protein down to 5 pg, with peaks of changing intensity (spectra labeled with area under the curve and signal to noise). The identities of these peaks were then confirmed by MS/MS. The areas under the curve for the peaks are plotted, showing detection in a linear range from 5 pg to 5 ng of protein.
FIG. 8 shows sample fractionation methods. The localization of TRX (spiked into whole plasma at a concentration of 2 ng/μL) was tracked through several different sample preparation procedures by western blotting (representative blots shown). Samples were precipitated by ETOH or by TCA, and equivalent portions of the supernatant (S) and pellet (P) analyzed (top). Samples denatured in several different ways were then also subjected to spin filtration (YM-50 Microcon filter), and equivalent portions of the eluent (E) and retentate (R) were analyzed (bottom). MALDI spectra showing the benefits of immunodepletion of keratin peptides from a sample.
FIGS. 9A-9F shows HPLC-MS detection of digested TRX and TRX 80. Elution times for nano-reverse phase LC were determined for the two peptides of interest. The identifying peptide for TRX 80 (914 KDa) consistently eluted at 25.75 minutes on a 60 minute linear gradient, while that for TRX (3484 KDa) eluted at 35.55 minutes. CNBr digests of recombinant TRX and TRX 80 were spiked into 100 μg of CNBr-digested whole human plasma, in consecutive tenfold dilutions. The mixture was then examined by LC-MS. Both peptides of interest were detected at levels down to 10 ng from within the complex plasma mixture (representative spectra shown, experiment performed in duplicate).
FIGS. 10A-10D shows the detection of TRX and TRX 80 by LC-MRM. All charge states of the two peptides of interest were identified, and MRM transitions were then developed and optimized for each parent ion on the 4000 QTRAP system. XICs obtained from an LC-MRM assay of 50 fmole of both heavy and light peptides are shown. Lower limits of quantitation (LLOQs) of 1-5 fmole and 500 amole were obtained for TRX and TRX 80, respectively in buffer. Conventional high-flow chromatography was used (R2/10 Poros column, AB).



DESCRIPTION
The present inventors describe herein a method in which samples (e.g. plasma or CSF) are digested (e.g. chemically digested using cyanogen bromide (CNBr)), resulting in the production of unique, identifying peptides for TRX and for TRX 80. Methods such as mass spectrometry analysis then can be used for the detection and quantitation of these peptides after digestion of a complex sample, eliminating the problems of antibody cross reaction, the necessity of available conformational epitopes for detection, and complications due to protein-protein interactions. The inventors detail a process of assay development to detect these proteins at biologically relevant levels, beginning with validating the chemical digestion protocol and examining the limits of detection for these peptides by MALDI in conjunction with various sample fractionation methods. In addition, they describe the development of a sensitive MRM assay for consistent, accurate quantitation of TRX and TRX 80 that has applications for research and clinical samples.
One aspect of the invention is a method for detecting a full-length protein and a truncated form (e.g., a naturally occurring cleavage product) thereof, in a sample, comprising
optionally denaturing and/or reducing proteins in the sample,
cleaving the proteins into smaller peptides, and
detecting a unique peptide identifier for the full-length protein and/or a unique peptide identifier for the truncated protein, in the sample.
In embodiments of this method, proteins in the sample are cleaved by chemical cleavage (e.g., cleavage at aspartyl residues by formic acid, cyanogen bromide cleavage, or 2-iodosobenzoic acid cleavage (IBA)) or with a protease (e.g., trypsin, chymotrypsin, or Lys-C). The chemical or enzymatic agents that are selected to cleave a protein of interest is a function of, for example, the sequence of the protein to be cleaved. The choice of an appropriate cleavage agent will be evident to a skilled worker. The agents indicated above are all useful for the cleavage of TRX and its variants. In embodiments of the invention, the N-terminal peptides from the full-length and the truncated protein are different, and can serve as unique peptide identifiers for the two proteins; or the C-terminal peptides from the full-length and the truncated protein are different, and can serve as unique peptide identifiers for the two proteins.
In one embodiment of the invention, the full-length protein is thioredoxin (TRX), and the truncated form thereof is its biologically active, C-terminal truncated, 10 kDa cleavage product, TRX 80. In this embodiment, the unique peptide identifier for TRX can be PTFQFFKKGQKVGEFSGANKEKLEATINELV (SEQ ID NO:1), and the unique peptide identifier for TRX 80 can be PTFQFFK (SEQ ID NO:2). In this method, the proteins in the sample are optionally denatured and reduced. The need for denaturation and/or reduction is a function of the type of cleavage being carried out. For example, denaturation is required for CNBr cleavage, but not for digestion with trypsin. It will be evident to a skilled worker if denaturation and/or reduction is required for a particular cleavage method. The proteins are then cleaved by chemical cleavage (e.g., as described above) or with a protease (e.g., as described above); the C-terminal peptides from the full-length and the truncated protein are different, and can serve as unique peptide identifiers for the two forms of the protein. In one embodiment, the proteins in the sample are denatured and reduced and are then cleaved with cyanogen bromide, to produce, among other peptides common to the two forms of the protein, the unique peptide identifiers represented by SEQ ID NO:1 and SEQ ID NO:2.
In methods of the invention, the unique peptide identifiers can be detected by any of a variety of methods, including HPLC, ELISA, electrochemiluminescence, flow cytometry based bead assays, mass spectrometry (MS), a multiple reaction monitoring assay (MRM), or selective reaction monitoring assay (SRM).
The detection step in a method of the invention can be quantitative.
The sample used in a method of the invention can be, e.g., a cell homogenate, a tissue homogenate, a biopsy tissue homogenate, serum/plasma, cerebrospinal fluid (CSF), synovial fluid, urine, cardiac tissue, tears, saliva, or culture medium in which cells have been grown.
Another aspect of the invention is an antibody that is specific for a contiguous sequence of between 5 and 7 amino acids that is shared by the peptides represented by SEQ ID NO:1 and SEQ ID NO:2. In one embodiment of the invention, the antibody is specific for the sequence, PTFQFFK (SEQ ID NO:2).
Another aspect of the invention is a composition comprising
a) an antibody specific for the peptide represented by SEQ ID NO:1, and/or
b) an antibody specific for the peptide represented by SEQ ID NO:2, and/or
c) an antibody specific for a peptide represented by a contiguous sequence of between 5 and 7 amino acids that is shared by SEQ ID NO:1 and SEQ ID NO:2.
Another aspect of the invention is a method for diagnosing a disease or condition in a subject, wherein the disease or condition is characterized by a level of TRX and/or TRX 80 that is altered by a statistically significant value compared to a control (e.g., a value that is proportional to the level in a subject that does not exhibit symptoms of the disease or condition), the method comprising detecting the presence and/or amounts of TRX and TRX 80 in a sample from the subject by the a method of the invention.
Another aspect of the invention is a kit for performing one of the methods of the invention. The kit can comprise, e.g., antibodies that are specific for one or more unique identifying peptides for distinguishing between two or more forms of one protein, or one or more unique identifying peptides for distinguishing between variants of more than one protein. A kit of the invention can be used, e.g., for pre-processing a sample for analysis by a method of the invention.
Any of a variety of proteins and variants thereof can be distinguished from one another by a method of the invention, provided that cleavage of a protein and a variant thereof results in at least one peptide that is unique to the protein (a unique peptide identifier for the protein) and at least one peptide that is unique to the variant (a unique peptide identifier for the truncated variant). As used herein, the terms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, as used above, more than one protein can be analyzed simultaneously by a method of the invention, and more than one variant can be distinguished for each of the proteins.
For example, a protein can be distinguished from a form of the protein that is truncated at its N-terminus and/or at its C-terminus. In a method of the invention, proteins in a sample are cleaved (e.g., digested) into smaller peptides. Cleavage conditions are selected so that, in addition to peptides that are in common between the protein and its variant, one or more peptides are generated that are unique to each form of the protein. In one embodiment of the invention, in which a full-length protein and a C-terminal truncated form thereof are distinguished, a sample comprising both forms of the protein is treated with a cleavage agent so that the C-terminal peptide of the full-length protein is longer than the C-terminal peptide of the truncated form.
A “unique peptide identifier” for a particular protein, as used herein, refers to a peptide which, following cleavage of the proteins in a sample, is generated from the particular protein, but not from a variant of that protein which an investigator wishes to distinguish from the particular protein, and which thus can be used to identify that protein. Furthermore, the unique peptide identifier must not be common to other cleaved proteins in the sample, which can comprise a complex mixture of proteins. For example, if the proteins of interest are in a human sample, the unique peptide identifier must not be generated by digestion of any other protein in that human sample. An investigator can insure that a peptide identifier is not present in other proteins in the sample being analyzed by performing a conventional BLAST analysis of the peptide identifier against, e.g., all known human proteins.
In another embodiment of the invention, instead of distinguishing a C-terminal truncated form of a protein from a version of the protein that is not truncated at its C-terminus, an N-terminal truncated form of a protein is distinguished from a version of the protein that is not truncated at its N-terminus. In this embodiment, the peptide from the N-terminus of the truncated protein is shorter than the N-terminal peptide of the non-truncated protein.
In other embodiments of the invention, proteins that differ from one another by internal sequences in the protein can be distinguished. For example, unique peptide identifiers can be used which correspond to internal deletions, or even single amino acid changes, within a variant form of the protein.
Much of the description herein is directed to the detection of the full-length form of TRX and of the truncated form thereof referred to as TRX 80, which is a biologically active cleavage form of TRX, having a molecular weight of 10 kDa and a truncation at the C-terminal end. However, it will be evident to a skilled worker that any pair (or set) of suitable proteins can be distinguished by a method of the invention. The description herein thus applies to such other pairs (or sets) of proteins, as well.
Furthermore, the naturally truncated form of TRX (TRX 80) that is discussed herein is the human form of the processed protein. However, the TRX protein can be differentially processed in other biological systems. Therefore, among the cleaved forms of TRX (e.g., in the form of isolated peptides) that can be identified by a method of the invention are:








PTFQFFKKGQKVGEFSGANKEKLEATINELV
(SEQ ID NO: 7)


 


PTFQFFKKGQKVGEFSGANKEKLEATINEL
(SEQ ID NO: 8)


 


PTFQFFKKGQKVGEFSGANKEKLEATINE
(SEQ ID NO: 9)


 


PTFQFFKKGQKVGEFSGANKEKLEATIN
(SEQ ID NO: 10)


 


PTFQFFKKGQKVGEFSGANKEKLEATI
(SEQ ID NO: 11)


 


PTFQFFKKGQKVGEFSGANKEKLEAT
(SEQ ID NO: 12)


 


PTFQFFKKGQKVGEFSGANKEKLEA
(SEQ ID NO: 13)


 


PTFQFFKKGQKVGEFSGANKEKLE
(SEQ ID NO: 14)


 


PTFQFFKKGQKVGEFSGANKEKL
(SEQ ID NO: 15)


 


PTFQFFKKGQKVGEFSGANKEK
(SEQ ID NO: 16)


 


PTFQFFKKGQKVGEFSGANKE
(SEQ ID NO: 17)


 


PTFQFFKKGQKVGEFSGANK
(SEQ ID NO: 18)


 


PTFQFFKKGQKVGEFSGAN
(SEQ ID NO: 19)


 


PTFQFFKKGQKVGEFSGA
(SEQ ID NO: 20)


 


PTFQFFKKGQKVGEFSG
(SEQ ID NO: 21)


 


PTFQFFKKGQKVGEFS
(SEQ ID NO: 22)


 


PTFQFFKKGQKVGEF
(SEQ ID NO: 23)


 


PTFQFFKKGQKVGE
(SEQ ID NO: 24)


 


PTFQFFKKGQKVG
(SEQ ID NO: 25)


 


PTFQFFKKGQKV
(SEQ ID NO: 26)


 


PTFQFFKKGQK
(SEQ ID NO: 27)


 


PTFQFFKKGQ
(SEQ ID NO: 28)


 


PTFQFFKKG
(SEQ ID NO: 29)


 


PTFQFFKK
(SEQ ID NO: 30)


 


PTFQF
(SEQ ID NO: 31)






A skilled worker will be able to identify which of the above forms of the peptides are present in a given biological system. A unique peptide identifier for TRX or a C-terminal deletion thereof can be any contiguous sequence from the N-terminus of the peptide beginning with PTFQ (SEQ ID NO:32) up to the full length of SEQ ID NO:1 without the C terminal valine (V).
Further, while the sequences above are specific to the mammalian form of TRX and TRX80, this method may be applied to other homologues of TRX and TRX80 in other species. A skilled worker would be able to identify which homologues or orthologues of TRX and TRX80 are present in an alternative species by using conventional BLAST comparisons.
The levels of TRX and/or TRX 80 appear to be a link between oxidative stress and inflammation. An assay capable of quantitating both the full-length and truncated forms of TRX is highly valuable. The present inventors describe herein an exemplary assay for distinguishing between, and quantitating, these two forms of the TRX protein, using both LC-MALDI and multiple reaction monitoring (MRM) mass spectrometry-based assays, in which digestion of samples with cyanogen bromide (CNBr) results in unique identifying peptides for both TRX and TRX 80 that can be quantitated in complex samples, including plasma. These methods overcome limitations of antibody-based detection methods that are imposed by the properties of TRX and TRX 80.
Samples to be analyzed by a method of the invention can originate from a variety of biological sources, including, e.g., humans or other animals, plants, viruses, etc. A variety of types of cells, tissues, organelles etc. can serve as sources for samples for a method of the invention. These include, e.g., serum/plasma, cerebral spinal fluid (CSF), synovial fluid, cell homogenates, tissue homogenates, urine, cardiac tissue, tears, saliva, biopsy tissues or the like.
A variety of conventional methods can be used to cleave proteins in a method of the invention. A cleavage method is selected which results in peptides, particularly unique peptide identifiers, that are of a size that can be readily resolved by the detection method to be used. For example, when mass spectrometry is used to analyze the resulting peptides, a cleavage method is selected which results in peptide fragments in an observable mass range for tandem MS mass spectrometers (about 500-about 7000 DA). When the peptides are detected by contact with an antibody, they should be of a size that is amenable to antibody binding. Such peptides can be, e.g., at least about 5-8 amino acids in length, e.g. about 5-20, or more, amino acids, or about 8-15 amino acids. As used herein, the term “about” refers to plus or minus 10%. For example, “about 8 amino acids” is 7-9 amino acids. A “range” of values, as used herein, includes the end points of the range. Thus, for example, 5-7 includes both 5 and 7.
In one embodiment of the invention, well-known chemical cleavage methods are employed, such as cleavage at aspartyl residues by formic acid, cyanogen bromide cleavage, 2-iodosobenzoic acid cleavage (IBA, e.g. using 2-Nitro-5-thiocyanatobenzoic acid powder), BNPS-Skatole cleavage, hydroxylamine cleavage, etc.
In another embodiment, a protease, generally a site-specific protease, or a combination of such proteases, is used. The proteases can be selected from, e.g., serine proteases, threonine proteases, cysteine proteases, aspartic acid proteases (e.g., plasmepsis), metailoproteases, glutamic acid proteases, or combinations thereof. Suitable proteases include, e.g., the proteases shown in Table 1.








TABLE 1








Achromopeptidase



Aminopeptidase



Ancrod



Angiotensin Converting Enzyme



Arg-C proteinase



Asp-N endopeptidase



Asp-N endopeptidase + N-terminal Glu



Bromelain



Calpain



Calpain I



Calpain II



Carboxypeptidase A



Carboxypeptidase B



Carboxypeptidase G



Carboxypeptidase P



Carboxypeptidase W



Carboxypeptidase Y



Caspase



Caspase 1



Caspase 2



Caspase 3



Caspase 4



Caspase 5



Caspase 6



Caspase 7



Caspase 8



Caspase 9



Caspase 10



Caspase 13



Cathepsin B



Cathepsin C



Cathepsin D



Cathepsin G



Cathepsin H



Cathepsin L



Chymopapain



Chymase



Chymotrypsin - high specificity



(C-term to FYW], not before P



Chymotrypsin - low specificity



(C-term to FYWML (SEQ ID



NO: 33)], not before P



Clostripain (Clostriiopeptidase B)



Collagenase



Complement C1r



Complement C1s



Complement Factor D



Complement factor I



Cucumisin



Dipeptidyl Peptidase IV



Elastase, leukocyte



Elastase, pancreatic



Endoproteinase Arg-C



Endoproteinase Asp-N



Endoproteinase Glu-C



Endoproteinase Lys-C



Enterokinase



Factor Xa



Ficin



Furin



Glutamyl endopeptidase



Granzyme A



Granzyme B



HIV Protease



IGase



Kallikrein tissue



Leucine Aminopeptidase (General)



Leucine aminopeptidase, cytosol



Leucine aminopeptidase microsomal



LysN



Matrix metalloprotease



Methionine Aminopeptidase



Neutrase



Papain



Pepsin (pH 1.3)



Pepsin (pH > 2)



Plasmin



Prolidase



Pronase E



Prostate Specific Antigen



Protease, Alkalophilic from



Streptomyces griseus



Protease from Aspergillus



Protease from Aspergillus saitoi



Protease from Aspergillus sojae



Protease (B. licheniformis) (Alkaline)



Protease (B. licheniformis) (Alcalase)



Protease from Bacillus polymyxa



Protease from Bacillus sp



Protease from Bacillus sp (Esperase)



Protease from Rhizopus sp.



Protease S



Proteasomes



Proteinase from Aspergillus oryzae



Proteinase 3



Proteinase A



Proteinase K



Protein C



Pyroglutamate aminopeptidase



Renin



Rennin



Staphylococcal peptidase I



Streptokinase



Subtilisin



Thermolysin



Thrombin



Tissue Plasminogen Activator



Tobacco etch virus protease



Trypsin



Tryptase



Urokinase









Among the commonly used proteases are: Endoproteinase Asp-N from a Pseudomonas fragi mutant; Endoproteinase Glu-C from Staphylococcus aureus V8; Endoproteinase Glu-C from Staphylococcus aureus V8; Endoproteinase Lys-C from Lysobacter enzymogenes; Endoproteinase Pro-C from E. coli BioChemika; Endoproteinase Pro-Pro-Y-Pro; Papain; Pepsin; Proteinase A (e.g. from S. cerevisiae); Proteinase K; Proteinase from Bacillus licheniformis Type VIII; α-Chymotrypsin; and Trypsin. See, e.g., the Sigma-Aldrich catalogue.
Combinations of chemical and protease methods of cleavage can also be used.
In some embodiments, proteins in a sample are denatured and/or reduced before they are cleaved. These procedures may be necessary for exposing desired cleavage sites to a cleavage agent, and/or for eliminating problems resulting from protein-protein interactions (either aggregation or, e.g., dimer formation of a particular protein, or interactions of a protein of interest with other proteins in the sample). A skilled worker will recognize in what situations denaturation and/or reduction of proteins in a sample may be beneficial. When quantitation of one or both unique peptide identifiers is performed, it is preferable that the cleavage (e.g., protease digestion or chemical cleavage) is carried out to completion.
A variety of types of methods can be used to detect unique peptide identifiers in a sample. The detection may be quantitative. For example, the peptides can be detected mass spectrometry. (sometimes abbreviated herein as “mass spec”). Among the many suitable detection methods are, e.g., HPLC; Western blotting (immunoblotting); ELISA; isoelectric focusing followed by Western blotting ELISA, or mass spec of fractions; antibody selection followed by immunological detection or mass spec (MS); matrix-assisted laser desorption/ionization spectroscopy (MALDI, or LC-MALDI); surface-enhanced laser desorption/ionization (SELDI), electrospray ionization mass spectroscopy (ESI); TOF; quadropole; ion trap; orbitotrap mass analyzers and various configurations of these (e.g., triple quad, etc); a multiple reaction monitoring assay (MRM); selective reaction monitoring assay (SRM); and multiple reaction monitoring (MRM).
In one embodiment of the invention, mass spectrometry is used. Among the variety of methods of mass spectral analysis that can be employed, which use different forms of ionization, are, e.g., electron ionization, chemical ionization (CI), electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), inductively coupled plasma (ICP), glow discharge, fast atom bombardment (FAB), thermospray, desorption/ionization on silicon (DIOS), direct analysis in real time (DART), atmospheric pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS), thermal ionization, nanospray, corona discharge, atmospheric pressure MALDI (AP-MALDI), desorption electrospray ionization (DESI), and chemical ionization (CI).
It will be evident to a skilled worker that a variety of methods of mass spectral analysis can be performed, using different forms of ionization. These include, e.g., electron ionization and chemical ionization for gases and vapors, and electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) for liquid and solid biological samples. A variety of sources can be used, including inductively coupled plasma sources, glow discharge, fast atom bombardment (FAB), thermospray, desorption/ionization on silicon (DIOS), direct analysis in real time (DART), atmospheric pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS), thermal ionization, nanospray, corona discharge, atmospheric pressure MALDI (AP-MALDI), desorption electrospray ionization (DESI), chemical ionization (CI) and inductively coupled plasma (ICP). Different sources of ionization can give rise to analytes (e.g. peptides) having different charges. For example, in ESI, peptides often exhibit multiple charges (e.g. +2H, +3H); whereas in MALDI, peptides almost exclusively have only a single charge. The manner in which analytes (e.g. peptides) receive their charge has an effect on the peptides that are observed. Some peptides ionize better by one method than the other, and vice versa. There is not 100% overlap between what is observed in MALDI vs. what is observed in ESI. In some cases, a peptide identified as being highly ionizing by one of these methods may not be observed with the other method. Therefore, it may be necessary in some cases to use antibodies against different peptides for MALDI as for ESI applications. However, because there is some overlap between targets identified by the ESI and MALDI methods, it may in some cases be possible to use the same antibodies for both MALDI and ESI applications. A skilled worker can readily determine which peptides (and antibodies thereto) are suitable for use for which type of ionization procedure. A method of the invention can be used to identify thioredoxin peptides for a variety of types of ionization, and a variety of types of mass spectrometry.
The detection (identification and, optionally, quantitation) of full-length thioredoxin (TRX) and/or its truncated product (TRX 80) can be used for laboratory assays or for clinical assays, in the context of many diseases, as well as general health. It can also be used for laboratory animal research or in the context of veterinary medicine. Clinically, TRX and/or TRX 80 may be diagnostic markers of disease on their own, or in combination with other markers. Varying ratios of the amount of TRX to its truncated form of TRX 80 in biological systems can also constitute valuable markers of disease processes.
The level of TRX is increased in the context of many disease processes, examples of which are well-known to those of skill in the art. Elevated levels appear to be a general marker of oxidative stress. Oxidative stress can lead to the increased expression and secretion of TRX, which then can be cleaved by monocyctes into a 10 kDa product, TRX 80. TRX 80 possesses monocyte chemoattractant activity, inducing monocyte differentiation and activation into a highly inflammatory phenotype termed the TRX80 activated monocyte (TAM). TRX 80 is detected at highly variable plasma levels of presumed healthy donors and has been implicated in rheumatoid arthritis. Levels of TRX/TRX80 appear to be a link between oxidative stress and inflammation.
Increased plasma levels of TRX have been demonstrated in many disease conditions, including, e.g., heart failure, cardiomyopathy, cancer, asthma, and rheumatoid arthritis, among others. Further, the ratio of amounts of TRX/TRX80 have been shown to be correlated with certain disease conditions, and with the progress of those conditions. Thus, tracking levels of TRX and/or TRX 80 can serve as a therapeutic or diagnostic marker for a variety of disease conditions, provide a valuable clinical assay for monitoring disease progression in many conditions, and aid in evaluating the efficacy of therapeutic interventions for certain diseases. Such assays can be used in conjunction with other diagnostic methods, including the detection of other biomarkers, for the disease state being analyzed.
In general, when performing a diagnostic assay for a disease condition in a subject, an investigator compares the levels of TRX and/or TRX 80 in the subject to a control value or negative reference standard (e.g. a level proportional to that in a subject that does not express symptoms of the disease condition). A value of TRX and/or TRX80 that is statistically significantly higher (or in some cases, lower) than that of the control is indicative of the presence of the disease condition. If desired, a positive reference standard, which correlates with the presence of the disease condition, can be used instead of, or in addition to, the negative standard. A “significantly” elevated or decreased level of TRX and/or TRX 80 (compared to a reference standard) is a level whose difference from the value of the reference standard is statistically significant, using statistical methods that are appropriate and well-known in the art, generally with a probability value of less than five percent chance of the change being due to random variation.
Using samples from subjects as a clinical diagnostic reference standard is generally not practical on a routine basis. Instead, one way to generate negative and positive reference standards is to use lysates from cells in culture, and establish a cut-point value by a direct comparison of the cell culture lysates to a true positive and true negative. Alternatively, reference standards (values) obtained from accumulated data or databases (e.g. published by others) can be used.
Exemplary disease conditions for which TRX appears to be a biomarker are shown in Table 2:







TABLE 2







Conditions in which thioredoxin has been proposed to play a role or is a biomarker


(partial list)









Disease/context
Thioredoxin:
References





Alzheimer's
Decreased TRX levels in patient brains
(Lovell et al, 2000)



TRX80 not examined



Rheumatoid
TRX80 Secreted by synovial cells in RA
(Lemarechal et al,


Arthritis
Elevated TRX levels in synovial fluid correlate
2007)



with inflammation
(Maurice et al, 1999)


Atherosclerosis
Increased plasma levels
(Miyamoto et al,



Increased expression in endothelial cells and
2005)



macrophages in plaques
(Takagi et al, 1998)



Role in pathogenesis is debated




TRX80 not examined



Asthma
Elevated serum TRX
(Yamada et al, 2003)



Application of exogenous TRX may ameliorate
(Ichiki et al, 2005)



oxidative stress in pulmonary inflammation




TRX80 not examined



Cancer
Over-expression of TRX in many solid tumors
(Grogan et al, 2000)



High levels of TRX in chemotherapy resistant
(Yamada et al, 1996)



tumors
(Shao et al, 2001)



Secretion of TRX from neoplastic cells
(Jordan et al, 2005)



PX-12 Thioredoxin inhibitor is in clinical trials




for therapy




TRX80 not examined



Cardiac
Protective antioxidant properties of TRX
(Ago and Sadoshima,


Hypertrophy
Also role of TRX as a growth factor,
2007)



contributing to hypertrophy
(Yamamoto et al,



TRX80 not examined
2003) (Sadoshima et




al, 2007)


Diabetes
Increased plasma TRX levels
(Miyamoto et al,



SNP in TXN UTR associated with susceptibility
2005) (Ikegami et al,



to Type I diabetes
2008) (Schulze et al,



TXNIP, inhibitor of TRX, is upregulated in
2004)



diabetes




TRX80 not examined



HIV
Increased plasma levels of TRX
(Nakamura et al,



Increased TRX levels in bone marrow
1996)



Depletion of TRX positive cells in lymph nodes
(Van Laer et al,



TRX reductase suppresses TAT dependent HIV
2002) (Masutani et



transcription
al, 1992) (Kalantari



TRX80 associated with elevated HIV replication
et al, 2008) (Newman



in Mφ in culture
et al, 1994)


Hypertension
Different models suggest increases or decreases
(Ebrahimian and



in expression
Touyz, 2008)



Decreased TRX expression in myocardium
(Tanito et al, 2004)



TRX80 not examined



Ischemia-
Exogenous TRX or TRX over-expression is
(Okubo et al, 1997)


Reperfusion
protective
(Takagi et al, 1998)



Increased TRX expression is seen in the brain
(Tao et al, 2006)



with ischemia




TRX80 not examined



Malaria
TRX system is essential for parasite to survive
(Becker et al, 2004)



host immune defense, TRX reductase has been




target for potential inhibitors




TRX80 not examined



HBV, HTLV-1,
Elevated TRX serum levels
(Sumida et al, 2000)


HCV
TRX80 not examined
(Masutani et al,




2005)









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In one embodiment of the invention, in which the sample is plasma, a specific CNBr cleaved peptide from hemoglobin can also be tracked and quantitated to indicate levels of hemolysis in the sample. This type of analysis can be useful for the development of a correction for the contribution of TRX by hemolysis, which can be an important consideration.
In one embodiment of the invention, an antibody is produced which is specific for a common region of the two unique TRX peptides discussed herein (e.g., PTFQFFK (SEQ ID NO:2), or a shorter version, such as a 5-6 amino acid version, of this sequence). This antibody can be used, e.g., for detection of the peptides, or to develop an affinity capture platform which would allow for the enrichment of these peptides prior to later detection methods such as mass spectrometry. Using such antibodies in this manner can readily allow for the simplification of sample peptide mixtures and the subsequent detection of TRX and TRX 80 at very low concentrations in samples of highly diverse protein composition. One of the key challenges in current mass spectrometry methods is that as sample complexity is increased, the ability to detect specific analytes (here two unique peptides) may be decreased. Currently, affinity based methods are often employed to reduce sample complexity by depleting samples such as plasma of high abundance proteins such as albumin. However, TRX can be found associated with albumin, and this would also deplete TRX from the samples. Thus, the ability to pre-process complete samples by chemical digestion, and to then affinity target the desired unique identifying peptides without concerns of protein interactions allows added efficiency of detection of the proteins at low levels.
The production of such an antibody can also enable the use of this digestion method in conjunction with gel based applications involving separation of the peptides based on size or isoelectric point followed by antibody based detection (e.g. immunoblotting), but with the elimination of the problems associated with protein interactions in samples, masking of epitopes, or the requirement for native conformation.
In addition, this method can be expanded to also include evaluation of protein modifications occurring on the unique or shared peptides of TRX and TRX 80 after digestion. This includes examination of the modification of the active site cysteines in the protein with oxidation, disulfide bond formation, nitrosylation or other mechanisms, and also such changes to other residues of the proteins, including cysteine modifications outside of the active site. These modifications can be detected with the use of mass spectrometry or an antibody raised to the specific modification.
An antibody that is “specific for” a peptide refers to an antibody that preferentially recognizes a defined sequence of amino acids, or epitope, that is present in the peptide, and not generally other peptides unintended for binding to the antibody. An antibody that “binds specifically” to (“is specific for”; binds “preferentially” to) a peptide of the invention interacts with the antibody, or forms or undergoes a physical association with it, in an amount and for a sufficient time to allow, e.g., the peptide to be removed from the solution, or to be captured from the solution, in conjunction with a method of the invention. By “specifically” or “preferentially” is meant that the antibody has a higher affinity, e.g. a higher degree of selectivity, for such a peptide than for other peptides in a sample. For example, the antibody can have an affinity for the peptide of at least about 5-fold higher than for other peptides in the sample. Typically this is application specific. For example, it does not matter if the antibody cross-reacts with peptides from proteins of different samples, if those peptides are not present in the sample of interest. The affinity or degree of specificity can be determined by a variety of routine procedures, including, e.g., competitive binding studies.
Methods for producing specific antibodies against a peptide of interest and for purifying the peptides or antibodies are conventional. The peptides used for generation of the antibodies can be produced by a variety of methods, including isolating them from purified proteins that have been cleaved with a suitable enzymatic or chemical method. Alternatively, the peptides can be produced using conventional chemical synthesis techniques, such as those described, e.g., in G. Barony et al., The Peptides: Analysis, Synthesis & Biology, Academic Press, pp. 3-285 (1980). Some chemically synthesized peptides can be obtained from commercial suppliers. Alternatively, a peptide of the invention can be produced recombinantly, using conventional genetic engineering techniques.
Generally, a peptide against which antibodies are to be produced is isolated or substantially purified before it is used to stimulate antibody formation. The term “substantially purified,” as used herein refers to a molecule, such as a peptide, that is substantially free of other proteins, peptides, lipids, carbohydrates, nucleic acids and other biological materials with which it is naturally associated. For example, a substantially pure compound, such as a peptide, can be at least about 60%, by dry weight, preferably at least about 70%, 80%, 90%, 95%, or 99% the molecule of interest. Methods for isolating (purifying) proteins or peptides are conventional.
An “antibody,” as used herein, can be, e.g., polyclonal, monoclonal (mAb), recombinant, humanized or partially humanized, chimeric, single chain, Fab, or fragments of such antibodies. Other specific binding partners, such as aptamers, can also be used. The antibody can be of any isotype, e.g., IgM, various IgG isotypes such as IgG1′ IgG2a, etc., and it can be from any animal species that produces antibodies, including goat, rabbit, mouse, chicken or the like. A mixture of antibody types can be used. It is noted that antibodies raised against purified peptides, even polyclonal antibodies, will exhibit high degrees of specificity for a cognate peptide.
Antibodies can be prepared according to conventional methods, which are well known in the art. See, e.g. Green et al., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), (Humana Press 1992); Coligan et al., in Current Protocols in Immunology, Sec. 2.4.1 (1992); Kohler & Milstein (1975), Nature 256, 495; Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Laboratory Pub. 1988). Methods of preparing humanized or partially humanized antibodies, antibody fragments, etc. and methods of purifying antibodies, are conventional.
Another aspect of the invention is a composition comprising two (or more) unique peptide identifiers for distinguishing two (or more) forms of a protein of interest. In one embodiment, the composition comprises unique peptide identifiers for distinguishing more than one protein and its variants. Such a composition can be used, e.g., to generate antibodies that are specific for the unique peptide identifiers. The antibodies may be of any of the types discussed herein, or combinations thereof.
Another aspect of the invention is a kit for carrying out any of the methods of the invention. For example, one embodiment is a kit for detecting the presence of unique peptide identifiers of interest in a sample. In one embodiment of the invention, a kit comprises one or more antibodies that are specific for one or more unique peptide identifiers and, optionally, means for storing or packaging the antibodies. The antibodies may be in a lyophilized form or in liquid form; they may be stabilized. Alternatively, a kit may comprises reagents for cleaving proteins in a sample, and/or for analyzing the resulting peptides.
The components of the kit will vary according to which method is being performed. Optionally, the kits comprise instructions (e.g., written instructions) for performing the method. Other optional elements of a kit of the invention include suitable buffers, media components, or the like; containers; or packaging materials. The reagents of the kit may be in containers in which the reagents are stable, e.g., in lyophilized form or stabilized liquids. The reagents may also be in single use form, e.g., in amounts for detecting unique peptide identifiers from a single sample, or for carrying out a single diagnostic test. Other optional elements of a kit include affinity chromatography columns in various sizes and configurations depending on the number of samples to be processed. The kit components are dependent on what type of detection method is used. For example, an ELISA kit might contain a capture antibody bound to a solid phase surface (e.g., plastic plate or bead) with detection antibodies able to distinguish either form of peptide, whereas a mass spectrometry based kit could include materials to render proteins into peptides, affinity purify the peptides and elute into a suitable medium for detection by mass spectrometry. Other components of a kit can easily be determined by one of skill in the art. Such components may include suitable controls or standards, buffers or other reagents appropriate for constituting a reaction medium allowing the formation of a peptide-antibody complex, etc.
Abbreviations used herein include the following:
















CNBr-
Cyanogen Bromide



CV-
coefficient of variance



Da-
Daltons



ESI-
electrospray ionization



ETOH-
ethanol



HPLC-
high performance liquid chromatography



KDa-
Kilo Daltons



LC-
liquid chromatography



LLOQ-
lower limits of quantitation



MALDI-
matrix assisted laser desorption ionization



MRM-
multiple reaction monitoring



MS-
Mass Spectrometry



MS/MS-
tandem mass spectrometry



NADPH-




RA-
rheumatoid arthritis



SCX-
strong cation exchange (chromatography)



TRX-
Thioredoxin



TRX80-
Truncated Thioredoxin



TAM-
TRX80 activated monocyte



TCA-
trichloro-acetic acid



TOF-
time of flight



XIC-
extracted ion chromatograph










In the foregoing and in the following examples, all temperatures are set forth in uncorrected degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.
EXAMPLES
Example I
Materials and Methods
Sample Material. Recombinant human TRX and TRX80 (R&D Systems) were resuspended in dH2O and aliquots were immediately digested or stored at −80° C. until used. Human plasma obtained from presumed healthy control donors was aliquoted immediately upon collection and stored at −80° C. Whole plasma was used for digestion, without any prior protein depletion. Undepleted pigtail macaque CSF samples used had been archived at −80° C. in aliquots from previous studies performed in a well-characterized SIV/macaque model (Zink et al. (2002) Journal of neurovirology 8 Suppl 2, 42-48; Clements et al. (20008) Journal of neurovirology 14, 309-317; Witwer et al. (2009) PLoS ONE 4(12), e8129)). For detection of TRX and TRX 80 in CSF, samples were obtained during the late stage of infection from macaques that were SIV-infected, SIV-infected and treated with the tetracycline derivative minocycline beginning at 12 or 21 days after SIV inoculation, or from uninfected control animals (Zink et al. (2005) Jama 293, 2003-2011; Follstaedt et al. (2008) Journal of neurovirology 14, 376-388)).
CNBr Digestion. CNBr digestion was performed by a protocol adapted from Samyn and colleagues (Samyn et al. (2005) Nat Methods 2, 193-200). Samples of either recombinant proteins, plasma, or CSF were first denatured and reduced with 10 mM dithiothreitol (DTT) in 7 M guanine hydrochloride (GuHCl), 0.3 M Tris, pH 9.0 for 45 minutes at 55° C. Iodoacetemide (55 mM in 200 mM NH4HCO3 [pH 7.0]) was added for alkylation, performed with shaking for 45 minutes in the dark at room temperature. Denatured, alkylated plasma or CSF samples were then precipitated with 20% TCA on ice for  ̃1 hr followed by a 15 min spin at 10,000×g (4° C.) to obtain a protein pellet. Alternately, recombinant protein samples (typically 5 ug) were desalted by applying to a Prosorb membrane (Applied Biosystems) and washing twice with 100 ul of dH2O. CNBr (10 M solution prepared fresh in acetonitrile, ACN; Sigma) was then added with deionized water and TFA (1:1:3 vol) to the protein pellet resulting from TCA precipitation (in the case of plasma and CSF) or to the protein adhered to a Prosorb membrane (for recombinant protein). TCA precipitated pellets were slowly solubilized with multiple additions of TFA and extensive vortexing before adding CNBr and dH2O. Digestion proceeded overnight at 4° C. in the dark with gentle shaking. After digestion of recombinant proteins on the Prosorb membrane, peptides were extracted in 70% ACN with 0.1% TFA from the membranes through two 30 min incubations at 37° C., then the pooled extracts were dried in a vacuum concentrator. Additional volumes of 70% ACN with 0.1% TFA also were added to the plasma and CSF digests to wash the samples, aiding in the removal of volatile TFA and CNBr during subsequent vacuum concentration.
Evaluation of CNBr Digests by SYPRO staining and Western Blotting. Samples were separated by electrophoresis on 16.5% or 10-20% gradient tris-tricine gels (BioRad). For total protein staining, gels were fixed during two 30 min incubations (50% methanol, 7% acetic acid) with gentle agitation. They were then stained with SYPRO Ruby (60 mL; Pierce) overnight with agitation at room temperature. Stained gels were washed twice for 30 min (10% methanol, 7% acetic acid), followed by a brief rehydration in dH2O. Imaging was performed on a Typhoon phosphorimager (excitation 528 nm, emission 610 BP; GE Healthcare). For western blotting, samples were run on 15% tris-HCl gels (BioRad) and transferred to PVDF membrane (Immobilon PSQ; Millipore). Membranes were blocked with 5% milk in TBST for ≧2 hrs at RT, followed by incubation in primary anti-TRX antibody in the same solution overnight at 4° C. (1 μg/mL; R&D Systems). After four washes with TBST, blots were then incubated with HRP conjugated secondary antibody (anti-goat-HRP, DAKO, 1:2000) for 1 hr at room temperature, followed by multiple washes with TBST and chemiluminescent detection using Super Signal West Dura substrate (Pierce).
MALDI Sample Preparation. Digested samples were cleaned over C18 columns prior to direct MALDI analysis or reverse phase separation. OMIX tips (Varian Inc.) or high capacity C18 columns (NEST) were used, dependent on the quantity of protein in a given sample. Digested peptides were re-suspended in 0.1% TFA prior to clean up, then applied to the columns/tips following manufacturers' instructions. After washing, peptides were eluted in 90% ACN+0.1% TFA. They were then dried in a vacuum concentrator before being re-suspended in 70% ACN+0.1% TFA for spotting to MALDI plates, or in 2% ACN, 0.1% TFA (solution A) in preparation for nano reverse-phase liquid chromatography.
MALDI-TOF/TOF Analysis. Samples (0.5 uL) were spotted to stainless steel MALDI plates with an equal volume of saturated alpha-cyano-4-hydroxy-cinnamic acid (CHCA; Sigma, recrystallized in house, or Ultrapure Matrix, Protea Biosciences) matrix solution in 50% ACN+0.1% TFA. 4700 Calibrant mixture (AB SCIEX) was used for plate calibration for all runs, with eight external calibrant spots monitored. Spectra of peptides were detected using an AB SCIEX TOF/TOF 4800 or 5800 system in positive ion mode MS with reflectron averaging 2500 shots. MS/MS was performed using a 2 KeV extraction method with CID turned off. Up to 5000 shots per peptide were obtained with auto quality setting set to stop acquisition once a total signal to noise of 70 is achieved.
HPLC Separations. A Tempo nano-LC system (AB SCIEX) was used for nano-reverse phase chromatography coupled with automated spotting of fractions for MALDI-TOF/TOF analysis. The spotting system consisted of an auto sampler, a two channel three pump true nano-flow liquid chromatography system (Eksigent Technologies) and an electrostatic spotting platform (AB SCIEX). Dried, C18 cleaned, digests of whole plasma with TRX and TRX 80 digests spiked in were re-suspended in Solution A (2% ACN, 0.1% TFA) and 3 μL samples (30 μg) auto injected onto a Merck Chromolith RP-18 column Peptides were separated on column using a 60 minute linear gradient from 2% ACN to 98% ACN at a flow rate of 4 μL/minute. Spotting of the eluted fractions mixed with alpha-cyano-4-hydroxy-cinnamic acid (CHCA) matrix (7 mg/mL in 70% ACN) to a MALDI plate was performed every 6000 ms throughout the gradient. SCX fractionation was performed by the Johns Hopkins Mass Spectrometry and Proteomics Facility using a PolyLC Inc. (Columbia, Md.) PolySULFOETHYL A column (10 cm×2.1 mm, 5 um particle size, 300 Angstrom pore size). 100 μg samples were re-suspended in 2 mL of loading buffer and loaded onto the column in two 1 mL injections followed by a 20 minute load equilibration time. Peptides were eluted at 250 μL/min in a 35 min gradient (3%-100% solvent B) using solvent A (10 mM potassium phosphate in 25% acetonitrile, pH 2.8) and solvent B (350 mM KCl in 10 mM potassium phosphate in 25% acetonitrile, pH 2.8). Absorbance was monitored and fractions were collected in one-minute intervals. Cleaning buffer was injected (1 mL) from 62-65 min and the column was then re-equilibrated for 20-30 min before beginning another fractionation. These fractions were subsequently dried down, and then desalted using C18 tips (OMIX) prior to spotting to a MALDI plate.
MRM Analysis. Isotopically labeled peptides incorporating heavy lysine residues were synthesized (*K; 13C6 15N2; New England Peptide) for use as quantitative standards for the two peptides of interest in an MRM assay. Each labeled residue added 8 Da to the peptide, for a total increase of 40 Da for the TRX peptide (PTFQFF*K*KGQ*KVGEFSGAN*KE*KLEATINELV (SEQ ID NO:34), 3525 Da), and 8 Da for the TRX80 peptide (PTFQFF*K (SEQ ID NO:35), 922 Da). MRM experiments were performed on a QqLIT triple quadrupole mass spectrometer (QTRAP® 5500 system or 4000 QTRAP®, AB SCIEX). MRMPilotTM software (AB SCIEX) was used to guide the initial development of MRM transitions. All charge states of the precursor ions were first determined, transitions were then chosen for each following optimization with changing collision energy. LC separation methods were developed for LC MRM using high flow conditions on a Shimadzu LC-20AD. Preliminary determination of limits of detection for the two peptides in buffer were performed with separation on a conventional R2/10 (2.1×100 mm) Poros column (Life Technologies), with a 5 min gradient from 5-95% ACN+0.1% formic acid. For detection of the peptides in the matrix of CNBR-digested plasma, various chromatography chemistries and methods were evaluated, and a Unison UK-C8 column (4.6 mm×150 mm) was found to give good resolution, as well as the potential for increased sample capacity. Transitions also were re-evaluated in the matrix of digested plasma and optimized for maximal detection without interference. A summed MRM transition was developed for TRX80, and three other transitions chosen to monitor TRX. Limits of quantitation (LOQ) were then determined for the two peptides of interest in plasma; lower limit of quantitation, LLOQ is defined by % CV<20% and accuracy of 80-120%.
Example II
Results
Diagnostic Peptides to Track and Quantitate TRX and TRX80
Using available protein sequence information for human TRX (NCBI), we determined that chemical cleavage of TRX and TRX80 with CNBr would yield two peptides common to both forms, but also would result in the creation of an additional, unique, identifying peptide for each (FIG. 1). This is not the case for the more regularly employed enzymatic method of tryptic digestion, for which all peptides produced from TRX80 also would be common to TRX. Searches for both of these identifying peptide sequences using BLASTp show that they are non-redundant to other known human protein sequences, making them unique identifiers (Altschul et al. (1997) Nucleic Acids Res 25, 3389-3402). Thus, a workflow in which a complex sample that contains TRX and/or TRX80 is reduced, alkylated, and then treated with CNBr can be used to produce a complex peptide mixture. These two unique peptides then can be tracked in this mixture to determine the amounts of TRX and/or TRX80 in the sample.
When samples of recombinant TRX or TRX80 were digested with CNBr, the proteins were neither detectable by western blot, nor were bands of the whole proteins (at approximately 12 and 10 kDa, respectively) visible in a total protein stained gel, both indicating that they were effectively digested (FIG. 6). Digestion of human plasma spiked with TRX and TRX80 (10 ng/μL) showed effective cleavage of these proteins in the matrix of plasma, as well as overall cleavage of total protein, illustrated by a loss of very high molecular weight species and an increase in low molecular weight polypeptides.
Following CNBr digestion of recombinant TRX and TRX80, peaks corresponding to the expected unique peptide masses were detected for each by MALDI (FIG. 1). Both peptides ionized efficiently, and were detectable from digests of consecutive tenfold dilutions of purified recombinant protein, from 50 ng to 5 pg, spotted directly to a MALDI plate and crystallized with alpha-cyano-4-hydroxy-cinnamic acid (CHCA) matrix (FIG. 7). The identities of these peptides were confirmed by obtaining MS/MS spectra and verified with theoretical masses for product ions.
Despite the very low absolute detection level attained by MALDI for these peptides, in the complex mixture of peptides produced from the digestion of whole human plasma, the peptides quickly become obscured, even at much higher levels. Attempts to overcome sample complexity and dynamic range issues, including specific protein depletion or enrichment methods and the use of crude fractionation of a total protein sample by molecular weight cut-off spin columns, were hindered due to strong interactions of TRX with other proteins. The use of ethanol precipitation to deplete an enriched albumin fraction (Colantonio et al. (2005) Proteomics 5, 3831-3835; Fu et al. (2005) Proteomics 5, 2656-2664) was found to be inappropriate in this study. Even with prior denaturation (with 10 mM DTT in 7 M GuHCl), TRX remained albumin-associated and was found in both fractions (FIG. 8). This suggests that other methods of albumin depletion may also remove TRX from the sample. TCA precipitation, while not useful for fractionating a sample, did successfully bring TRX into a protein pellet. We also attempted several methods of simplifying samples with spin columns Even using a relatively high molecular weight cut-off spin filtration unit far above the size of our proteins of interest (Microcon YM-50; Millipore), we were unable to get the majority of TRX spiked into test plasma samples (at 2 ng/μL) to successfully elute in a low molecular weight plasma protein fraction, even when samples were denatured with GuHCL, Urea, or ACN. A majority of the TRX protein remained in the high molecular weight retentate under the conditions we examined (FIG. 8). Because of this, we determined that denaturing and digestion of the complete complex sample to obtain smaller peptides prior to performing fractionation would be necessary for accurate quantitation.
Sample Fractionation by Nano-reverse Phase LC and Strong Cation Exchange (SCX)
Using nano-reverse phase HPLC for separation after protein digestion, we determined elution times for the two identifying CNBr peptides on a 60 min linear gradient (approximately 25.75 and 36.55 min for TRX80 and TRX, respectively). We then were able to detect these peptides at low levels when CNBr-digested recombinant TRX and TRX80 were spiked into CNBr digests of whole human plasma, down to a limit of detection (LOD) of 10 ng/100 μg plasma protein (FIG. 9). Fractionation increased detection of the desired peptides by MALDI due to ionization suppression by other abundant species. The LOD was, however, still higher than that obtained for MALDI analysis of digests of the recombinant proteins alone, and higher than that which would be predicted as necessary to detect both proteins in plasma samples under most biological conditions.
SCX fractionation separation of samples also was examined. When separated with a 35 min gradient from 0-350 mM KCl, the peptides for TRX80 and TRX consistently eluted in very narrow windows (centered on 9 and 34 minutes into the gradient, respectively). Samples with decreasing levels of digested recombinant TRX and TRX80 spiked into CNBr-digested plasma were fractionated, and for each sample several fractions surrounding these elutions were evaluated by MALDI. By this method, TRX was consistently detected down to a level of 10 ng/100 ug plasma. A slight gain in sensitivity was obtained for TRX80, with MS/MS verified detection at 1 ng/100 ug plasma (FIG. 2). Ultimately, a new MS platform for a more robust, sensitive assay based on the same CNBr digestion concept was necessary. We developed an MRM assay on a hybrid triple quadrupole/linear ion trap instrument.
MRM Assay for TRX and TRX80
To develop an assay for absolute quantitation, labeled standard peptides containing heavy lysine (*K; 13C6 15N2) were produced (FIG. 1). The charge states for heavy (quantitative standard) and light (endogenous) versions of both peptides were determined and their fragmentation spectra obtained on a 4000 QTRAP system. Three MRM transitions then were selected from the fragmentation data for each of these parent ions, optimized for maximal intensity under variable collision energies (FIG. 10). The assay development was performed by MRMPilot software, including some transitions for the larger TRX peptide that were manually optimized. An LC method was optimized for the effective retention and separation of these peptides for LC-MRM. High flow chromatography was used in order to develop a more robust method compared to nano-flow; such a method could then more readily be adapted for high throughput analysis of large sample sets in the lab or clinic. Additionally, the greater column capacity makes it possible for more total sample to be run, if necessary, to detect low-level analytes.
Using these methods on the 4000 QTRAP instrument, limits of quantitation for both TRX and TRX80 by LC-MRM in buffer were determined. The TRX80 peptide was detected to a limit of  ̃500 amoles, while TRX was detected at  ̃1-5 fmoles on column. This suggested that absolute detection of levels in the targeted range of low nanograms of the proteins is possible. However, detection in the matrix of plasma once again resulted in a higher level of background and potential for suppression.
To obtain a gain in sensitivity during optimization of a method for detection of these two peptides in CNBr-digested plasma, this work was migrated to the QTRAP 5500 system. The transitions developed were examined in the context of plasma, and re-optimized to account for matrix effects. Several different approaches to chromatography also were examined, and a Unison UK-C8 column (4.6 mm×150 mm) was found to provide optimal binding and separation of the CNBr plasma digest. LC-MRM was performed and transitions were monitored for dilutions of heavy standard peptides in CNBr-digested plasma to obtain standard concentration curves (dilutions of 1-10,000 fmole peptide in 2 μL of plasma protein). Two MRM transitions were monitored for the TRX80 peptide, and it was determined that the best sensitivity was obtained by summing the MRM transitions together for quantitation. Three MRM transitions also were used for TRX and these were used separately. When detection curves were produced in triplicate, lower limits of quantitation (LLOQ, defined by CV<20% and accuracy between 80-120%) were identified at 10 and 2 fmole for TRX and TRX80, respectively, in 2 uL CNBr-digested plasma. This is equivalent to detection of the proteins in plasma at levels of 59 ng/mL TRX and 9 ng/mL TRX80. XICs are shown for the LLOQ (FIG. 3).
Assaying TRX and TRX80 in Macaque CSF by MRM
Given that previously reported biological range of TRX in plasma extends slightly below the LLOQ reached in this assay, we decided to evaluate the assay in a matrix with lower interference. We chose to examine CSF, where TRX and TRX80 might provide insight into inflammatory CNS disease processes. As protein concentration and variation is generally far lower in CSF than plasma, we hypothesized that we would readily be able to detect TRX or TRX80 in CSF.
Since TRX levels have been shown to be elevated peripherally in HIV infection (Nakamura et al. (1996) Int Immunol 8, 603-611), we asked whether TRX might also be present in the CSF of SIV-infected macaques with encephalitis (Follstaedt et al. (2008) (supra)). The macaque TRX protein sequence is highly conserved with the human protein; the sequences of the two peptides of interest were identical. Detection curves were produced using pooled, CNBr-digested CSF samples as a matrix. LLOQs for the two peptides were identified at 1 fmole/μL for TRX and 0.5 fmole/μL for TRX80 (FIG. 4).
Seventeen individual macaque CSF samples were evaluated using this MRM assay: four uninfected control animals, eight SIV-infected untreated animals, and five SIV-infected animals treated with the drug minocycline as a potential anti-HIV, neuroprotective therapeutic (Zink et al. (2005) (supra)). Individual CNBr-digested CSF samples were spiked with heavy labeled reference peptides prior to assaying in triplicate. While TRX was identified at varying levels in all samples, the presence of TRX80 was not identified in any sample. It is possible that TRX80 levels in these samples fall <LOD for the assay. While there were no significant differences observed in the levels of TRX between minocycline-treated and untreated animals (not shown), when all animals were grouped by their encephalitis status, a significant difference existed between SIV-infected animals that developed no/mild encephalitis versus animals that developed moderate/severe encephalitis (FIG. 5, P=0.05, Mann-Whitney).
Discussion
In this study we demonstrated that CNBr digestion of samples containing TRX produces identifying peptides that can be used to monitor both TRX and TRX80. Standard protein fractionation methods are not effective for concentrating TRX in a simplified fraction due to the presence of the protein in multiple fractions. Thus, separation at the peptide level is necessary to prevent sample preparation biases. Using a peptide-level separation and identification method provided several advantages, bypassing protein-protein interaction concerns that can interfere in accurate quantitation and protein fractionation problems.
Although TRX and TRX80 were detectable by MALDI, even with RP-LC or SCX separation prior to MS, the sensitivity necessary for plasma assay was not attainable on this platform. Given the complex mixture of peptides produced from the digestion of whole human plasma, the target peptides quickly become suppressed, even when present at much higher abundance. MALDI based methods are expected to provide an effective assay for other samples such as synovial fluid or cerebrospinal fluid, which have decreased complexity. Given our goal of quantitating TRX and TRX80 in plasma, the move to a new MS platform for a more advanced, robust, sensitive assay based on the same CNBr digestion concept was necessary. We began development of an MRM assay on a hybrid triple quadrupole/linear ion trap instrument.
The goal of distinguishing TRX from TRX80 imposed the unique restriction on this MRM assay of pre-defining specific peptides to be examined for each form of the protein, as all transitions for each form had to be obtained from its single respective parent ion. Extensive validation was performed to show a solid MRM assay for quantitation, and multiple transitions were used for each single peptide. A recent study (Fortin et al. (2009) Mol Cell Proteomics 8, 1006-15) gave proof-of-principle for quantitation under such conditions, demonstrating the ability to develop an accurate, precise assay based on only one peptide after extensive assay validation to quantitate PSA to low ng/mL levels in serum using high flow LC.
In this study, LODs 100-times lower than those obtained by MALDI were reached by MRM, allowing for quantitation of levels in the ng/mL range (LLOQs 59 ng/mL TRX, 9 ng/mL TRX80). This is within the range observed in human plasma (Pekkari et al. (2000) J Biol Chem 275, 37474-37480; Baker et al. (2006) J Lab Clin Med 147, 83-90). The QTRAP 5500 system permits sensitive detection in plasma, and provides the option of further developing a multiple reaction monitoring cubed (MRM3) assay (Fortin et al. (2009) Anal Chem 81, 9343-9352) for higher specificity. The use of high-flow chromatography conditions in conjunction with the assay also permits increased sample capacity, if necessary. Digestion with CNBr makes this option very cost effective in contrast to the more expensive enzymatic digestion of large volume samples. High-flow chromatography also is a robust method that will allow this LC-MRM assay to be adapted for high-throughput use and multiplex quantitation with other biomarkers.
The biology of the TRX/TRX80 system is of broad interest to many different areas of research. TRX has been shown to play a role in many disease processes, and these continue to be very active areas of study. Elevated levels of secreted TRX in disease may provide some degree of protection from damaging oxidative radicals by way of its antioxidant properties. Administration of exogenous TRX as a potential therapeutic strategy has been evaluated in models of myocardial ischemia-reperfusion injury, cerebral ischemia, interstitial lung disease, chronic obstructive pulmonary disease, and others as a potential therapeutic strategy (Burke-Gaffney et al. (2005) Trends Pharmacol Sci 26, 398-404; Kinoshita et al. (2007) Biochem Biophys Res Commun 354, 712-719). However, its ability to protect against oxidative stress could be confounded by its potential to contribute to inflammatory immune processes, particularly if conditions favor its cleavage to TRX80. In many of the disease conditions in which there is elevated secreted TRX, it still is unclear whether it is contributing further to the pathology by affecting cell signaling processes, or is a response to oxidative stress of other disease processes, or possibly both. To elucidate TRX's potentially protective and harmful properties, the ability to quantitatively track TRX and TRX80 in biological systems is important.
Little is known of the roles of TRX and TRX80 in HIV infection. Plasma levels of TRX increase with progressing HIV infection, correlating with increased disease severity and lowered CD4+ T cell counts (Nakamura et al. (1996) Int Immunol 8, 603-611). TRX80 levels have not been examined in HIV infection, although an in vitro study showed that addition of exogenous TRX80 enhances HIV replication in macrophage cultures (Newman et al. (1994) J Exp Med 180, 359-363.) TRX could be an important component in modulating the phenotype and activation of peripheral monocytes and T cells in HIV infection. Activated cells of the monocyte lineage play a key role in the development of HIV-associated organ-specific diseases, such as CNS disease. Here we investigated TRX levels in CSF samples from an SIV/macaque model of HIV-associated neurological disease. TRX was detectable in CSF by this method, and secreted TRX was significantly higher in SIV-infected animals that develop moderate/severe encephalitis than in animals that develop no/mild encephalitis.
We were unable to convincingly identify TRX80 in these samples. There are several potential reasons for this, including the timing at which the samples were obtained during the disease model, the possibility that the cleavage site is not truly at the predicted 80 AA, or that TRX80 may not be present in the CSF under the conditions studied. Evaluation of samples collected longitudinally during disease progression should provide insight into oxidative stress and inflammatory processes, and help evaluate potential therapeutics for HIV CNS disease.
Understanding basic TRX related processes can shed light onto the regulation of oxidative stress both in health and in disease. This continually growing area of research will benefit from tools such as this MRM assay to monitor the proteins involved in an accurate, sensitive way.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make changes and modifications of the invention to adapt it to various usage and conditions and to utilize the present invention to its fullest extent. The preceding preferred specific embodiments are to be construed as merely illustrative, and not limiting of the scope of the invention in any way whatsoever. The entire disclosure of all applications, patents, and publications cited above, particularly with regard to the specific finding for which they are referenced herein, and in the figures, are hereby incorporated in their entirety by reference.

## Claims

1. A method for detecting a full-length protein and a truncated form thereof, in a sample, comprising the steps of:
optionally denaturing and/or reducing proteins in the sample;
cleaving the proteins into smaller peptides; and
detecting a unique peptide identifier for the full-length protein and/or a unique peptide identifier for the truncated protein, in the sample, wherein the full length protein is thioredoxin (TRX), and the truncated protein is its biologically active, C-terminal truncated, 10 kDa cleavage product, TRX 80, and wherein the unique peptide identifier for TRX is PTFQFFKKGQKVGEFSGANKEKLEATINELV (SEQ ID NO:1), and the unique peptide identifier for TRX 80 is PTFQFFK (SEQ ID NO:2).

2. The method of claim 1, wherein proteins in the sample are cleaved by a chemical cleavage method or with a protease, and the N-terminal peptides from the full-length and the truncated protein are different, and can serve as unique peptide identifiers for the two proteins.

3. The method of claim 2, wherein the chemical cleavage method is cleavage at aspartyl residues by formic acid, cyanogen bromide cleavage, or 2-iodosobenzoic acid cleavage (IBA).

4. The method of claim 2, wherein the protease is trypsin, chymotrypsin, or Lys-C.

5. The method of claim 1, wherein proteins in the sample are cleaved by a chemical cleavage method or with a protease, and the C-terminal peptides from the full-length and the truncated protein are different, and can serve as unique peptide identifiers for the two proteins.

6. The method of claim 5, wherein the chemical cleavage method is cleavage at aspartyl residues by formic acid, cyanogen bromide cleavage, or 2-iodosobenzoic acid cleavage (IBA).

7. The method of claim 5, wherein the protease is trypsin, chymotrypsin, or Lys-C.

8. The method of claim 1, wherein proteins in the sample are optionally denatured and reduced and are then cleaved by chemical cleavage or with a protease, and the C-terminal peptides from the full-length and the truncated protein are different, and can serve as unique peptide identifiers for the two forms of the protein.

9. The method of claim 8, wherein proteins in the sample are denatured and reduced and are then cleaved with cyanogen bromide, to produce, among other peptides common to the two forms of the protein, the unique peptide identifiers represented by SEQ ID NO:1 and SEQ ID NO:2.

10. The method of claim 1, wherein the unique peptide identifiers are detected by HPLC, ELISA, electrochemiluminescence, flow cytometry, based bead assays, mass spectrometry (MS), a multiple reaction monitoring assay (MRM), or selective reaction monitoring assay (SRM).

11. The method of claim 1, wherein the unique peptide identifiers are detected by mass spectrometry.

12. The method of claim 1, which is quantitative.

13. The method of claim 1, wherein the sample is a cell homogenate, a tissue homogenate, a biopsy tissue homogenate, serum/plasma, cerebrospinal fluid (CSF), synovial fluid, urine, cardiac tissue, tears, saliva, or culture medium in which cells have been grown.

14. A kit for carrying out the method of claim 1.