Toward a Human Blood Serum Proteome

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Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3–5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease. Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3–5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease. Serum, derived from plasma with clotting factors removed, contains 60–80 mg of protein/ml in addition to various small molecules including salts, lipids, amino acids, and sugars (1.Burtis C.A. Ashwood E.R. Tietz Fundamentals of Clinical Chemistry. 5th Ed. W. B. Saunders Company, Philadelphia, PA2001Google Scholar). The major protein constituents of serum include albumin, immunoglobulins, transferrin, haptoglobin, and lipoproteins (1.Burtis C.A. Ashwood E.R. Tietz Fundamentals of Clinical Chemistry. 5th Ed. W. B. Saunders Company, Philadelphia, PA2001Google Scholar, 2.Turner M.W. Hulme B. The Plasma Proteins: An Introduction. Pitman Medical & Scientific Publishing Co., Ltd., London1970Google Scholar). In addition to these major constituents, serum also contains many other proteins that are synthesized and secreted, shed, or lost from cells and tissues throughout the body (3.Schrader M. Schulz-Knappe P. Peptidomics technologies for human body fluids.Trends Biotechnol. 2001; 19: S55-S60Abstract Full Text Full Text PDF Google Scholar, 4.Kennedy S. Proteomic profiling from human samples: the body fluid alternative.Toxicol. Lett. 2001; 120: 379-384Google Scholar). It is estimated that up to 10,000 proteins may be commonly present in serum, most of which would be present at very low relative abundances (5.Wrotnowski C. The future of plasma proteins.Genet. Eng. News. 1998; 18: 14Google Scholar). Historically, two-dimensional PAGE has been the primary method of separation and comparison for complex protein mixtures. This method has been critical in developing our understanding of the complexity and variety of proteins contained in cells and bodily fluids. Two-dimensional PAGE has been used to analyze serum and plasma (the unclotted parent fluid of serum) (6.Eberini I. Agnello D. Miller I. Villa P. Fratelli M. Ghezzi P. Gemeiner M. Chan J. Aebersold R. Gianazza E. Proteins of rat serum V: adjuvant arthritis and its modulation by nonsteroidal anti-inflammatory drugs.Electrophoresis. 2000; 21: 2170-2179Google Scholar, 7.Eberini I. Miller I. Zancan V. Bolego C. Puglisi L. Gemeiner M. Gianazza E. Proteins of rat serum IV. Time-course of acute-phase protein expression and its modulation by indomethacine.Electrophoresis. 1999; 20: 846-853Google Scholar, 8.Haynes P. Miller I. Aebersold R. Gemeiner M. Eberini I. Lovati M.R. Manzoni C. Vignati M. Gianazza E. Proteins of rat serum: I. establishing a reference two-dimensional electrophoresis map by immunodetection and microbore high performance liquid chromatography-electrospray mass spectrometry.Electrophoresis. 1998; 19: 1484-1492Google Scholar, 9.Edwards J.J. Anderson N.G. Nance S.L. Anderson N.L. Red cell proteins. I. two-dimensional mapping of human erythrocyte lysate proteins.Blood. 1979; 53: 1121-1132Google Scholar, 10.Anderson 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, 11.Miller I. Haynes P. Gemeiner M. Aebersold R. Manzoni C. Lovati M.R. Vignati M. Eberini I. Gianazza E. Proteins of rat serum: II. influence of some biological parameters of the two-dimensional electrophoresis pattern.Electrophoresis. 1998; 19: 1493-1500Google Scholar, 12.Miller I. Haynes P. Eberini I. Gemeiner M. Aebersold R. Gianazza E. Proteins of rat serum: III. gender-related differences in protein concentration under baseline conditions and upon experimental inflammation as evaluated by two-dimensional electrophoresis.Electrophoresis. 1999; 20: 836-845Google Scholar, 13.Peters Jr., T. Intracellular precursor forms of plasma proteins: their functions and possible occurrence in plasma.Clin. Chem. 1987; 33: 1317-1325Google Scholar). Although impressive improvements in two-dimensional PAGE technologies have occurred in recent years, limitations remain. Two-dimensional PAGE is labor-intensive, requires relatively large sample quantities, is poorly reproducible, has a limited dynamic range for protein detection, and has difficulties in detecting proteins with extremes in molecular mass and isoelectric point (14.Rabilloud T. Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains.Proteomics. 2002; 2: 3-10Google Scholar). To address these limitations several types of mass spectrometry, in conjunction with various separation and analysis methods, are increasingly being adopted for proteomic measurements (15.Conrads T.P. Alving K. Veenstra T.D. Belov M.E. Anderson G.A. Anderson D.J. Lipton M.S. Pasa-Tolic L. Udseth H.R. Chrisler W.B. Thrall B.D. Smith R.D. Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling.Anal. Chem. 2001; 73: 2132-2139Google Scholar, 16.Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates III, J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Google Scholar, 17.Raida M. Schulz-Knappe P. Heine G. Forssmann W.G. Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate.J. Am. Soc. Mass Spectrom. 1999; 10: 45-54Google Scholar, 18.Liotta L.A. Kohn E.C. Petricoin E.F. 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A. 1977; 74: 5421-5425Google Scholar, 11.Miller I. Haynes P. Gemeiner M. Aebersold R. Manzoni C. Lovati M.R. Vignati M. Eberini I. Gianazza E. Proteins of rat serum: II. influence of some biological parameters of the two-dimensional electrophoresis pattern.Electrophoresis. 1998; 19: 1493-1500Google Scholar, 17.Raida M. Schulz-Knappe P. Heine G. Forssmann W.G. Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate.J. Am. Soc. Mass Spectrom. 1999; 10: 45-54Google Scholar, M.S. Proteomic analysis of human of to and other high molecular 2001; Scholar). The of this or separation is also by that many proteins as biomarkers for and and prostate-specific are a that is at least orders of the most serum proteins (1.Burtis C.A. Ashwood E.R. Tietz Fundamentals of Clinical Chemistry. 5th Ed. W. B. Saunders Company, Philadelphia, PA2001Google Scholar). the dynamic range by proteomic are to allow for of these lower abundance serum or without or separation of the high abundance proteins. with protein separation is that low abundance proteins may be removed with the and Ed. Scholar). is a protein of very high abundance in serum that would be a for prior to a proteomic analysis of lower abundance proteins. is a protein in blood serum that a large variety of including and amino (1.Burtis C.A. Ashwood E.R. Tietz Fundamentals of Clinical Chemistry. 5th Ed. W. B. Saunders Company, Philadelphia, PA2001Google Scholar, Proteins in Clinical Ed. for Blood Scholar, E. 5th Ed. Scholar). of from serum may also in the of low abundance and lipoproteins of or are also proteins in serum that by in blood and their To this variety of present in immunoglobulins (1.Burtis C.A. Ashwood E.R. Tietz Fundamentals of Clinical Chemistry. 5th Ed. W. B. Saunders Company, Philadelphia, PA2001Google Scholar, Proteins in Clinical Ed. for Blood Scholar, N.L. Anderson N.G. The human plasma and 2002; Scholar). are a of in serum that protein from with immunoglobulins and the of from their of immunoglobulins is for a proteomic analysis of The of this to to or high abundance serum proteins and to proteomic that increase the dynamic range for the identification and of serum proteins. include the of protein to to immunoglobulins, as a of in these include the separation of peptides prior to mass spectrometric analysis using strong cation exchange used strong cation liquid mass mass for used strong cation liquid mass mass for chromatography and reversed-phase liquid This a large number of proteins from a serum sample and the foundation for future with The human blood serum from a plasma from blood without by The plasma to at and the removed by under at our the serum into and at In proteins were and were using gel electrophoresis with at with a protein using serum as a protein and Ed. Scholar, and method for the of quantities of protein the of Scholar). The immunoglobulins were by affinity chromatography using protein of serum with an of and to protein that been with This with for at serum separated from the protein by The were with of and the were with the The serum sample into digested with for at and serum peptides were in of with The sample to and separated using an ion exchange of a with a of with and The and for with B. were using a of over and the at for an at a of The at with a and a of fractions were at using a fractions were and at for reversed-phase analysis. separation performed with an high liquid chromatography with a with of and and of and fractions were in of were the in at a of and the with for were with a from to over The to an ion trap mass using electrospray The mass to the with the of by a by the most precursor by mass in from the parent mass were used and from to 9 In for were with an range of and which contained high were using of and mass range also mass that removed to the most peptides that were in the analysis. mass were by SEQUEST A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates III, J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Google Scholar, III, J.R. Carmack E. L. A.J. Eng protein identification using liquid mass 1999; Scholar, III, J.R. Eng with Chem. Scholar, D. Yates III, J.R. analysis of the by protein identification Biotechnol. 2001; 19: Scholar, Yates J.R. An to of peptides with in a protein Am. Soc. Mass Spectrom. which its by mass with mass from a of protein are with and from the from the and these were to the possible for from the from to be as The mass and the ion SEQUEST analysis performed using a of the human protein with SEQUEST the of proteins and the of some protein as as the number of for serum proteins Jr., T. Intracellular precursor forms of plasma proteins: their functions and possible occurrence in plasma.Clin. Chem. 1987; 33: 1317-1325Google Scholar). were using the for human protein and the human to serum proteins were from our of the various human databases the of SEQUEST The of the databases in an in of the SEQUEST in a of peptides in the including visual of and in the of the analysis up to to a human protein has been and is to for a number of A. M. M. of the and the in 2002; Scholar). the to be an for this blood serum analysis to the and with a proteomic is for to with of To address the for we to analyze using a very peptides with to human proteins but still the of complexity in a derived from the of a (15.Conrads T.P. Alving K. Veenstra T.D. Belov M.E. Anderson G.A. Anderson D.J. Lipton M.S. Pasa-Tolic L. Udseth H.R. Chrisler W.B. Thrall B.D. Smith R.D. Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling.Anal. Chem. 2001; 73: 2132-2139Google used to SEQUEST to compare the human fractions and that contained the number of peptides were the D. for this SEQUEST were with to by Yates and D. Yates III, J.R. analysis of the by protein identification Biotechnol. 2001; 19: Scholar, Yates III, J.R. An protein identification for Chem. 2001; 73: Scholar). proteins in are by and R. Schulz-Knappe P. M. L. M. Forssmann W.G. of the in human blood of human Sci. 1999; Scholar). used to the serum the SEQUEST to allow for identification of peptides from and The and were derived by the peptides to the identified The high abundance and of a reference for The were that resulted in or for resulted in of the parameters for SEQUEST in a The parameters used to into the of the and the as in I. a of used for that SEQUEST to its and for identification Yates J.R. An to of peptides with in a protein Am. Soc. Mass Spectrom. Scholar). or peptides for an individual protein the in the mass for peptides were performed using as for assessment of A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates III, J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Google Scholar, Yates III, J.R. An protein identification for Chem. 2001; 73: Scholar). the be with the to be used in the the some be present the or for or amino is to be the an be as being or the mass of the precursor with or of the amino We that protein affinity chromatography of the immunoglobulins from serum as by electrophoresis of serum by is by the that proteins of proteins. In the serum immunoglobulins have of high that may an analysis of to increase the dynamic concentration range and of it is critical to the immunoglobulins from the serum sample. The and of the immunoglobulins were removed with is also by the This is in that the of the protein the from protein removed Scholar). and other proteins may also present for an analysis. of separation have resulted in or of low abundance proteins of in plasma M.S. Proteomic analysis of human of to and other high molecular 2001; or in fluid from M. Schulz-Knappe P. Heine G. Forssmann W.G. Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate.J. Am. Soc. Mass Spectrom. 1999; 10: 45-54Google Scholar, R. Schulz-Knappe P. M. L. M. Forssmann W.G. of the in human blood of human Sci. 1999; Scholar). from the serum, the used to peptides by and perform a separation with reversed-phase The chromatography resulted in with the over fractions The the of fractions to increase the number of proteins by an analysis. and were by using a for the most commonly peptides from the previous analysis. In by the to or for in The resulted in the number of peptides SEQUEST and as the analysis but resulted in proteins identified by peptides and of serum This increase in identification is to the analysis novel peptides high abundance peptides In for in dynamic range and in individual analysis we that some fractions may be complex to to the a analysis to and by in by in in I. in a Using and microcapillary reversed-phase high performance by analysis with SEQUEST we have identified 490 proteins in proteins include in III. Proteins in this analysis also a large concentration range from reference from with peptides from concentration with peptides from concentration with peptides from concentration R. P. of plasma and plasma concentration of and 2002; with from prostate-specific concentration in a (1.Burtis C.A. Ashwood E.R. Tietz Fundamentals of Clinical Chemistry. 5th Ed. W. B. Saunders Company, Philadelphia, PA2001Google Scholar). analysis most serum proteins as as a large number of proteins identified in serum P. Miller I. Aebersold R. Gemeiner M. Eberini I. Lovati M.R. Manzoni C. Vignati M. Gianazza E. Proteins of rat serum: I. establishing a reference two-dimensional electrophoresis map by immunodetection and microbore high performance liquid chromatography-electrospray mass spectrometry.Electrophoresis. 1998; 19: 1484-1492Google Scholar, 9.Edwards J.J. Anderson N.G. Nance S.L. Anderson N.L. Red cell proteins. I. two-dimensional mapping of human erythrocyte lysate proteins.Blood. 1979; 53: 1121-1132Google Scholar, 10.Anderson 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, 11.Miller I. Haynes P. Gemeiner M. Aebersold R. Manzoni C. Lovati M.R. Vignati M. Eberini I. Gianazza E. Proteins of rat serum: II. influence of some biological parameters of the two-dimensional electrophoresis pattern.Electrophoresis. 1998; 19: 1493-1500Google Scholar, 12.Miller I. Haynes P. Eberini I. Gemeiner M. Aebersold R. Gianazza E. Proteins of rat serum: III. gender-related differences in protein concentration under baseline conditions and upon experimental inflammation as evaluated by two-dimensional electrophoresis.Electrophoresis. 1999; 20: 836-845Google Scholar, R. Schulz-Knappe P. M. L. M. Forssmann W.G. of the in human blood of human Sci. 1999; Scholar, C. K. D. D. of proteins by mass spectrometry separation and Chem. proteins from the 490 proteins blood haptoglobin, serum proteins and and low proteins and blood and factors and factors and and growth proteins and protein protein and high protein growth protein protein and human growth hormone, protein growth prostate-specific and protein and and protein and growth in a SEQUEST analysis are using a combination of and in is the of the a and an experimental of An is for is the in the with the and the with the for is is in protein peptides from the protein that have and or to P. Miller I. Aebersold R. Gemeiner M. Eberini I. Lovati M.R. Manzoni C. Vignati M. Gianazza E. Proteins of rat serum: I. establishing a reference two-dimensional electrophoresis map by immunodetection and microbore high performance liquid chromatography-electrospray mass spectrometry.Electrophoresis. 1998; 19: 1484-1492Google Scholar, 16.Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates III, J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Google Scholar, Yates III, J.R. An protein identification for Chem. 2001; 73: Scholar). To the from a SEQUEST analysis, we the human peptides by and using SEQUEST with different The databases with SEQUEST analysis were an bacterial and a human protein The of from a SEQUEST analysis with the D. a of that is of low for a SEQUEST analysis using a human a of peptides with The the and high contains many the SEQUEST analysis of peptides using the D. but or of the low In or of the peptides from the human The method in for the using the human protein at a it is that most of the peptides identified from the D. that the as proteins serum, some of these peptides by or be using the D. Blood has many high abundance proteins that perform various Blood plasma contains or low abundance proteins that are critical for and and of cells may be into the The of these in blood the of using a proteomic for biomarkers for In this study, we an analysis of serum 490 proteins and at least a 3–5-fold increase in the number of identified proteins from a fluid in previous reports. proteomic of human plasma have used two-dimensional as the of Anderson and 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, C. L. A. R.D. The has the 1999; have been by the on-line human plasma two-dimensional PAGE previous have plasma and are to the serum of the proteins identified in this on-line human plasma protein we identified in our serum analysis. are several possible for these including and plasma but serum the clotting factors and our serum from a The concentration of blood proteins may for our sample a an is which is at in a (1.Burtis C.A. Ashwood E.R. Tietz Fundamentals of Clinical Chemistry. 5th Ed. W. B. Saunders Company, Philadelphia, PA2001Google Scholar). the sample and analytical used by these previous from The of for the other as may be to of sample and analysis. our is for identification the two-dimensional PAGE is up of but identified we the 490 including that would be to be of for comparison is the of rat serum by Gianazza and (6.Eberini I. Agnello D. Miller I. Villa P. Fratelli M. Ghezzi P. Gemeiner M. Chan J. Aebersold R. Gianazza E. Proteins of rat serum V: adjuvant arthritis and its modulation by nonsteroidal anti-inflammatory drugs.Electrophoresis. 2000; 21: 2170-2179Google Scholar, 7.Eberini I. Miller I. Zancan V. Bolego C. Puglisi L. Gemeiner M. Gianazza E. Proteins of rat serum IV. Time-course of acute-phase protein expression and its modulation by indomethacine.Electrophoresis. 1999; 20: 846-853Google Scholar, 8.Haynes P. Miller I. Aebersold R. Gemeiner M. Eberini I. Lovati M.R. Manzoni C. Vignati M. Gianazza E. Proteins of rat serum: I. establishing a reference two-dimensional electrophoresis map by immunodetection and microbore high performance liquid chromatography-electrospray mass spectrometry.Electrophoresis. 1998; 19: 1484-1492Google Scholar, 11.Miller I. Haynes P. Gemeiner M. Aebersold R. Manzoni C. Lovati M.R. Vignati M. Eberini I. Gianazza E. Proteins of rat serum: II. influence of some biological parameters of the two-dimensional electrophoresis pattern.Electrophoresis. 1998; 19: 1493-1500Google Scholar, 12.Miller I. Haynes P. Eberini I. Gemeiner M. Aebersold R. Gianazza E. Proteins of rat serum: III. gender-related differences in protein concentration under baseline conditions and upon experimental inflammation as evaluated by two-dimensional electrophoresis.Electrophoresis. 1999; 20: 836-845Google Scholar). identified proteins with human and the in protein abundance with or with disease. rat serum that proteins be for identified the human of of the identified rat proteins. We the human of or of the for a of with the plasma two-dimensional PAGE In differences may proteins and expression is a complex biological fluid with many and the or concentration of a protein may be the serum is In an analysis of this it is to that from for many proteins. of are and which are in the blood high and of blood cells it may be that and be in serum A. V. J. C. of a protein in the of J. Scholar). In to our peptides and peptides were In and are and from the serum the serum proteins and A. V. J. C. of a protein in the of J. Scholar, P. of and Scholar, R. of in of from the in the Scholar, C. K. P. of the and of plasma protein Scholar). of are the identification of evaluated by It is these peptides from of immunoglobulins in or from immunoglobulins in proteomic is an to and large with improvements in individual A. S. S. D.R. E. protein for mass 2002; Scholar, Yates III, J.R. of protein using a SEQUEST Chem. 2002; 74: Scholar, D. a that mass analysis, protein and in a 2002; 2: Scholar). proteomic are with individual with In a recent using a to proteins by using with blood serum has been to have in E.F. C. Kohn E.C. L.A. of proteomic in serum to 2002; Scholar). of the difficulties to the of SEQUEST for is the of to the of and the of to by by our SEQUEST to we have an and adopted method for in the of the may be and many plasma proteins are Jr., T. Intracellular precursor forms of plasma proteins: their functions and possible occurrence in plasma.Clin. Chem. 1987; 33: 1317-1325Google Scholar). factors include as and are that peptides with this variety and number of The serum proteins in this were identified from improvements to sample and are to these protein to the complexity of a proteomic analysis of serum Jr., T. Intracellular precursor forms of plasma proteins: their functions and possible occurrence in plasma.Clin. Chem. 1987; 33: 1317-1325Google Scholar). we to in and into the concentration range of proteins at least 9 orders of magnitude. improvements in the sample and separation with in the dynamic and to from mass spectrometry are to the beyond the 490 proteins identified in this the of human protein databases analysis are to be protein in this range that have been to human protein A. M. M. of the and the in 2002; Scholar). The has been to and future in human S. J. The human a to 2002; Scholar). the many of the of are the of the human proteomes and the of and with the of for analysis S. J. The human a to 2002; Scholar). we a large for proteomic analysis of this analysis 490 a protein of we have a method that be used to the of a SEQUEST proteomic analysis with the to protein for a SEQUEST analysis.