High Throughput Quantitative Analysis of Serum Proteins Using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry

Abstract

It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates. It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates. There is growing interest in testing the hypothesis that the serum 1In this paper, the term serum is used to indicate serum or plasma. proteome contains protein biomarkers that are useful for classifying the physiological or pathological status of an individual. Such markers are expected to be useful for the prediction, detection, and diagnosis of disease as well as to follow the efficacy, toxicology, and side effects of drug treatment (1Wulfkuhle J.D. Liotta L.A. Petricoin E.F. Proteomic applications for the early detection of cancer..Nat. Rev. Cancer. 2003; 3: 267-275Google Scholar). The idea of reading diagnostic or prognostic signatures from human body fluids is neither new nor original. Early attempts using high resolution two-dimensional gel electrophoresis were described more than 2 decades ago (2Anderson L. Anderson N.G. High resolution two-dimensional electrophoresis of human plasma proteins..Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5421-5425Google Scholar, 3Merril C.R. Goldman D. Sedman S.A. Ebert M.H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins..Science. 1981; 211: 1437-1438Google Scholar, 4Merril C.R. Switzer R.C. Van Keuren M.L. Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain..Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4335-4339Google Scholar). Renewed interest in this idea has emerged due to recent advances in proteomic technologies (5Aebersold R. Mann M. Mass spectrometry-based proteomics..Nature. 2003; 422: 198-207Google Scholar), intriguing initial results from analyzing serum protein patterns using mass spectrometry (1Wulfkuhle J.D. Liotta L.A. Petricoin E.F. Proteomic applications for the early detection of cancer..Nat. Rev. Cancer. 2003; 3: 267-275Google Scholar), and the clinical validation and use of a number of diagnostic disease markers including CA125 for ovarian cancer, prostate-specific antigen for prostate cancer, and carcinoembryonic antigen for colon, breast, pancreatic, and lung cancer (6Diamandis E.P. Mass spectrometry as a diagnostic and a cancer biomarker discovery tool: opportunities and potential limitations..Mol. Cell. Proteomics. 2004; 3: 367-378Google Scholar). A number of new approaches that differ from the traditional two-dimensional gel electrophoresis method for the discovery of protein biomarkers in serum have recently been described (1Wulfkuhle J.D. Liotta L.A. Petricoin E.F. Proteomic applications for the early detection of cancer..Nat. Rev. Cancer. 2003; 3: 267-275Google Scholar). These include surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS) 2The abbreviations used are: SELDI, surface-enhanced laser desorption ionization; MS, mass spectrometry; LC, liquid chromatography; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; MALDI, matrix-assisted laser desorption ionization; CID, collision-induced dissociation; TOF, time-of-flight; QTOF, quadrupole time-of-flight; CV, coefficient of variance; DMBA, 7,12-dimethylbenz[a]anthracene; HPLC, high performance liquid chromatography. (7Petricoin E.F. Ardekani A.M. Hitt B.A. Levine P.J. Fusaro V.A. Steinberg S.M. Mills G.B. Simone C. Fishman D.A. Kohn E.C. Liotta L.A. Use of proteomic patterns in serum to identify ovarian cancer..Lancet. 2002; 359: 572-577Google Scholar), liquid chromatography tandem mass spectrometry (LC-MS/MS) of serum proteome digests (8Adkins J.N. Varnum S.M. Auberry K.J. Moore R.J. Angell N.H. Smith R.D. Springer D.L. Pounds J.G. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry..Mol. Cell. Proteomics. 2002; 1: 947-955Google Scholar, 9Tirumalai R.S. Chan K.C. Prieto D.A. Issaq H.J. Conrads T.P. Veenstra T.D. Characterization of the low molecular weight human serum proteome..Mol. Cell. Proteomics. 2003; 2: 1096-1103Google Scholar, 10Shen Y. Jacobs J.M. Camp II, D.G. Fang R. Moore R.J. Smith R.D. Xiao W. Davis R.W. Tompkins R.G. Ultra-high-efficiency strong for high of the human plasma 2004; 76: Scholar), or protein separation by S. liquid in Sci. 2003; Scholar, S. approaches to the of biomarkers for 2002; Scholar), of the serum proteome on by matrix-assisted laser desorption ionization mass spectrometry D. E.C. peptide by sample and mass 2004; 76: Scholar), and and of these of the serum proteome is with the of serum samples. human blood serum is to of of of protein that a of an of Anderson N.G. The human plasma proteome: and diagnostic Cell. Proteomics. 2002; 1: Scholar). the serum proteome is by a few highly proteins, the human serum proteins of total protein mass R.S. Chan K.C. Prieto D.A. Issaq H.J. Conrads T.P. Veenstra T.D. Characterization of the low molecular weight human serum proteome..Mol. Cell. Proteomics. 2003; 2: 1096-1103Google Scholar). of total serum protein mass is by protein, of the serum proteins show two-dimensional that are with the of protein Anderson N.G. The human plasma proteome: and diagnostic Cell. Proteomics. 2002; 1: Scholar). protein from two-dimensional of serum were by mass spectrometry, to protein on were as of the R. C.R. A.M. M. R. Anderson S. The human serum proteome: of protein on two-dimensional electrophoresis gels and of 2003; 3: Scholar). the serum proteome in an and in a for serum proteome analysis have the to detect low quantitative to in the proteome and to detect in a of to the to identify peptides for their on and of results from and and high sample throughput to support with statistical Here we a new method for quantitative serum proteome It is on the selective isolation of peptides from serum proteins that are N-linked glycosylated in the protein and the analysis of the peptide the now deglycosylated of these peptides by and By selectively this of the a in analyte complexity a of the total number of peptides due to the that serum protein on contains a few N-linked and a of complexity by the that significantly to the peptide We on to show that this method is and increased and throughput compared with the analysis of selective analyte we in a peptide patterns the serum proteome of mice from genetically identical untreated normal mice be and peptides be this method of the and high throughput of the serum We that in serum discovery were from from from were from mice of were to the skin L.A. A. A. of or of skin 74: Scholar). were were and were with The of mice were with a of the in of were with a for to that were well of with as early as and to for the A of these to mice were and blood by with a and to for were by The untreated mice the mice as by N-linked glycosylated peptides were and using the N-linked as described R. and of N-linked using and mass 2003; Scholar). from of serum were used in isolation and of formerly N-linked and peptides from of serum were used in mass spectrometry tryptic peptides from serum proteins, proteins from of serum were in of for The proteins were with of and the proteins were The peptides were reduced by for and by for The peptides were and in from of serum of serum were used for The peptides and proteins were using analysis using an mass as described S.A. M.H. R. analysis of protein using Scholar). quantitative analysis of peptides using an mass peptides from of serum sample using the method were a peptide with using a and a with The from the a electrospray ionization in peptides were and the mass The peptide a high for ESI, and the were to of a of E.C. R. A liquid chromatography electrospray ionization of peptide tandem mass spectrometry the level on an mass with Mass 2003; Scholar). A of from a of the data were with a in the mass between and with and of the peptide mass were from data analysis The were used for analysis for with the of samples. data a of or to data for this and be C. and R. in The use data by analysis of formerly N-linked glycopeptides from serum and the to peptides that are of in cancer and normal A of peptide from The this a of a for the analysis of data A. R. proteomic analysis by mass 2003; Scholar). to the that in peptides are in were the to detected in patterns were on peptide The used to peptides with the The for in the in peptide by the the significantly the sample the high mass in the and the that highly and peptide patterns. the peptides that in of in were for further quantitative of peptides in for peptide using the method as described in the for data R. statistical analysis of protein from data by and tandem mass 2003; Scholar). the from of a peptide and and for peptide the for of the peptide were The a level in and that from the the The of peptides with their were to unsupervised clustering D. analysis and of Natl. Acad. Sci. U. S. A. to identify peptides cancer from normal samples. to the data were to and the of peptide in of the total were used for clustering The of the method is the of peptide patterns the serum to the detection of peptides that between of and the of these The method is in and consists of that N-linked in the protein were in their deglycosylated using a recently described method R. and of N-linked using and mass 2003; Scholar). peptides were by to generate and patterns. patterns from were and the peptides were peptides and the proteins from were by tandem mass spectrometry and data the of the method for serum protein serum from genetically identical were using the N-linked and the peptides were by The resulting collision-induced were the data and the data results were further using the A. R. statistical to the of peptide by and 2002; 74: Scholar). of the in peptide from the data with peptide of to a of A. R. statistical to the of peptide by and 2002; 74: The were for the of the N-linked The number of proteins by the peptides were using R. of proteins using and mass Scholar). 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We are of a of the protein between the human and serum the serum two-dimensional of human and are to an of the of the proteins in this between human and A. M.L. A serum two-dimensional gel to and 2004; Scholar). the proteins proteins are to be in human serum low These include and II, and for and serum of the proteins in have been in the two-dimensional that are low in serum A. M.L. A serum two-dimensional gel to and 2004; Scholar). the detection the of the peptides from these proteins were using the of the the used for peptide of the an peptide of is than the for these experiments that multidimensional serum proteins on the of be detected by of formerly N-linked of formerly N-linked glycopeptides from sera and the of their proteins in human N-linked peptide between the are by plasma to and to N-linked peptide between the are by to in a new the peptides and proteins by peptides and proteins were from The number of peptides in is low compared with the total number of peptides We used the D. E.C. R. A to and data by liquid 2004; 76: to these were due to peptide in the or the data in the is as the patterns of the peptides from serum were peptides by due to the complexity of the peptides in a analysis were for and for are with and peptides from these are with or on the of peptide as as be from the between the number of peptides from analysis and total peptides in a sample from a of the high peptides from were selectively by the between by between were by the that a of total peptides by analysis in the of peptides in in of the are by these results that of glycopeptides from genetically identical mice are using due to peptide results in a number of peptide and a of the We the of the peptide patterns by from a serum sample were in to generate and by to we of and the sample by using a were used to detect in the resulting to and to peptide between patterns. 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Scholar). nor about the of normal The results of this unsupervised clustering analysis are by the A of the peptides a of peptides is in in a peptide and a serum The of the are to the of the peptide patterns. this is that the mice and were and from the patterns from their and normal mice and were the serum be the we tryptic peptides from of serum sample to the and analysis were from the resulting and a number of peptide were detected as for the samples. to the unsupervised clustering of the total serum peptide patterns the cancer from the normal These results indicate that the number of proteins the the serum proteome by the is to the between serum to the clinical state of the The were further by to identify peptides that in in mice compared with untreated normal The and of these peptides were to the on a tandem mass and by and data shows a peptide of variation between consistently increased in mice and compared with normal and The of as a peptide with the the formerly N-linked from serum in is an protein is to be elevated Anderson and analysis of serum Scholar). We further the of the peptides by quantitative analysis using these the of the glycopeptides were with and the peptides were to the support their isolation R. and of N-linked using and mass 2003; Scholar). of from mice and and normal mice and were with the and and the peptides were in the sample with sample sample with sample sample with sample and sample with sample The were by The of peptides with in mice using analysis and were and the for and peptides were in the mass a for the of and a for the by analysis using and by data the peptides and proteins with elevated protein level in the detected by analysis and by The are in on The for the peptide from serum is in The increased level of this peptide in mice by with that by analysis these data indicate that the analysis of formerly N-linked glycosylated peptides detected peptides of in serum of cancer and normal mice and that the peptides be by of peptides and proteins with elevated in N-linked peptide between the are by number in on N-linked peptide between the are by in a new We a method for high throughput quantitative analysis of serum proteins using and It consists of the selective and isolation of peptides from the serum proteome that are by N-linked in the intact The of the deglycosylated of these peptides by The mass of peptides using and these peptides were by results indicate that the method is for the isolation of N-linked peptides were per protein an of per is with a tryptic peptides per protein from the of proteins. The data indicate that this reduced sample complexity in an in sensitivity compared with the analysis of serum digests using an identical for analysis of the method to the of sera from genetically identical mice that were untreated normal or The resulting peptide patterns and be via unsupervised of the peptides were further by MS/MS, and their in cancer control mice by using for the detection and validation of protein biomarkers in the serum of clinical be and to the complexity of the serum proteome and the proteomic technologies for can sample a of the the proteins Anderson N.G. The human plasma proteome: and diagnostic Cell. Proteomics. 2002; 1: Scholar, W. R. and tandem mass a for the quantitative analysis of and 2004; Scholar). two-dimensional gel have about serum proteins Anderson N.G. The human plasma proteome: and diagnostic Cell. Proteomics. 2002; 1: Scholar, R. C.R. A.M. M. R. Anderson S. The human serum proteome: of protein on two-dimensional electrophoresis gels and of 2003; 3: Scholar, M. R. R.S. Conrads T.P. Veenstra T.D. J.N. Pounds J.G. R. A. The human plasma proteome: a by of Cell. Proteomics. 2004; 3: Scholar). It has been that approaches have detection of low proteins due to the high of serum proteins and the of the (6Diamandis E.P. Mass spectrometry as a diagnostic and a cancer biomarker discovery tool: opportunities and potential limitations..Mol. Cell. Proteomics. 2004; 3: 367-378Google Scholar). the method the selective isolation of the N-linked glycosylated peptides in a in the of protein detected due to the in sample A number of to this the number of peptides per protein the method is significantly The proteins in this are to generate an of tryptic peptides per on the N-linked and can be and an of peptides N-linked per protein were By a number of N-linked per protein by and Y. M. and mass spectrometry to identify N-linked 2003; per in a in N-linked glycopeptides were from proteins using the serum protein, N-linked and is to the of total serum protein of peptides that serum peptide samples. quantitative of a that is by use of R. Anderson S. an a of the human plasma 2003; 3: Scholar), is an of the the method peptides from the of and the number of is of total protein mass in serum The and and a of an Anderson N.G. The human plasma proteome: and diagnostic Cell. Proteomics. 2002; 1: Scholar). The of the of in serum proteome recently in a in a tryptic of serum by strong the plasma protein were Y. Jacobs J.M. Camp II, D.G. Fang R. Moore R.J. Smith R.D. Xiao W. Davis R.W. Tompkins R.G. Ultra-high-efficiency strong for high of the human plasma 2004; 76: Scholar). It is that an more of peptides in patterns of serum protein digests are from and protein data the of to serum proteins are by and resulting in for It has been that protein generate on the of Anderson N.G. The human plasma proteome: and diagnostic Cell. Proteomics. 2002; 1: Scholar). the of the are the complexity of the peptide The peptides by the method the and by a few peptides per protein of The of these is the of a peptide sample from the serum proteome with a of an of peptides per an of potential N-linked glycopeptides an is for the serum proteins of these potential N-linked were of these potential N-linked be S.M. analysis of the protein of for and 2004; Scholar), the peptides from be by mass spectrometry, or protein be by the protein as the the number of peptides from increased due to of protein and in the It is expected that the to an of the number of peptides digests of serum were of peptides from of serum using the we were to detect and peptide that were of in with a of peptides were due to the complexity of the sample and the that the mass to a of the the highly peptides in sample The the of the of protein using this we used for quantitative and this to the peptide in including from proteins of low the of peptide is for of the proteome per is that to the of proteins are in this is that the of proteins are in glycosylated and blood and as markers for diagnosis and Scholar), proteins that a of biomarkers serum the of peptides per protein the of the this in protein level or level markers that protein including be detected on a peptides by a potential disease markers that are due to and blood and as markers for diagnosis and Scholar). this we used the and analysis to serum from mice with skin cancer from that of littermates. this the mice with skin cancer and their untreated the and in the The a with skin cancer the The sera were by the of consistently increased or between the cancer and control in this the low number of to detect the of the method to of potential biomarkers in more human the analysis of sample to statistical validation of the The has throughput to a few a number that to generate results a M. R. J.D. M.L. M. M. Y. of biomarker for early detection of Natl. Scholar, Y. Davis Y. protein coupled with a prostate cancer from prostate and 2002; Scholar). By a to sample and by further analysis and the of a data analysis we are further the performance of the to the used and the detected in the method are molecular peptides in between and These for in a tandem mass are By the of peptides to an we have serum proteins for the is increased in with the of skin cancer in mice these proteins are of and have been to the in of cancer S. C. S. D. R. C. from cancer a protein composition Cell. Proteomics. 2002; 1: Scholar), are markers for the diagnosis of skin useful for cancer detection, or be proteins in from the of a of the to the or in the serum the detection of proteins or patterns of proteins, is that are that potential markers or signatures in and can be and the proteomic biomarker discovery to molecular signatures as the of and to between biomarkers and The of peptides in this that some of the proteins in in the skin cancer are to highly serum cancer markers in clinical use have in the (6Diamandis E.P. Mass spectrometry as a diagnostic and a cancer biomarker discovery tool: opportunities and potential limitations..Mol. Cell. Proteomics. 2004; 3: 367-378Google has that the method and by are about of from the sensitivity to detect proteins. The method has the potential to sensitivity and high performance are used a of of serum sample contains of prostate-specific an that is detected in a mass serum digests are on the the total of serum that can be to the be and the of detection be reduced compared with the prostate-specific antigen be well the detection of an further in the of detection were the method be with peptide including electrophoresis or chromatography or selectively peptides from N-linked glycosylated serum proteins has been to be a method for the analysis of the serum with the high of this the high level of serum proteome a throughput that this method be useful for the detection of proteins or protein patterns that in physiological We and for We and for with and analyzing the data from the serum samples.