Evaluation of Large Scale Quantitative Proteomic Assay Development Using Peptide Affinity-based Mass Spectrometry

Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays. Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays. Highly specific and sensitive assays (e.g. immunoassays) are not available for quantifying the vast majority of human proteins, and de novo assay generation is associated with a high cost and long lead time. Consequently, although genomic and proteomic technologies are used to routinely identify many hundreds of candidate biomarkers for a given disease, very few undergo further verification and validation, which require a quantitative assay. This conundrum is likely a major contributing factor to the highly inefficient translation of candidate biomarkers into clinical use (1Paulovich A.G. Whiteaker J.R. Hoofnagle A.N. Wang P. 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Multiple reaction monitoring mass spectrometry (MRM-MS) 1The abbreviations used are:MRMmultiple reaction monitoringSISCAPAstable isotope standards and capture by antipeptide antibodiesQCquality control. has been used for decades in clinical reference laboratories for accurate quantitation of small molecules in plasma, such as drug metabolites or metabolites that accumulate as a result of inborn errors of metabolism (4Chace D.H. Kalas T.A. A biochemical perspective on the use of tandem mass spectrometry for newborn screening and clinical testing.Clin. Biochem. 2005; 38: 296-309Crossref PubMed Scopus (132) Google Scholar, 5Want E.J. Cravatt B.F. Siuzdak G. The expanding role of mass spectrometry in metabolite profiling and characterization.Chembiochem. 2005; 6: 1941-1951Crossref PubMed Scopus (197) Google Scholar). More recently, MRM-MS has been adapted to measure the concentrations of candidate protein biomarkers in plasma and cell lysates (6Barr J.R. Maggio V.L. Patterson Jr., D.G. 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One or more selected peptides whose sequence is unique to the target protein in that species (i.e. “proteotypic” peptides) are then measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution methods (i.e. a spiked-in stable isotope labeled peptide standard), MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices (12Lange V. Picotti P. Domon B. Aebersold R. Selected reaction monitoring for quantitative proteomics: A tutorial.Mol. Syst. Biol. 2008; 4: 222Crossref PubMed Scopus (1131) Google Scholar, 13Pan S. Aebersold R. Chen R. Rush J. Goodlett D.R. McIntosh M.W. Zhang J. Brentnall T.A. Mass spectrometry based targeted protein quantification: Methods and applications.J. Proteome Res. 2009; 8: 787-797Crossref PubMed Scopus (314) Google Scholar). The assays are specific, precise (%CV ≤ 20%) (14Agger S.A. Marney L.C. Hoofnagle A.N. Simultaneous quantification of apolipoprotein A-I and apolipoprotein B by liquid Chromatography–Multiple reaction Monitoring/Mass spectrometry.Clin. Chem. 2010; 56: 1804-1813Crossref PubMed Scopus (132) Google Scholar), multiplex-able (15Picotti P. Bodenmiller B. Mueller L.N. Domon B. Aebersold R. Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics.Cell. 2009; 138: 795-806Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar), and portable across laboratories and instrument platforms (16Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. Spiegelman C.H. Zimmerman L.J. Ham A.J. Keshishian H. Hall S.C. S. C.H. T. J. M. T.A. H. Smith L.J. S. Wang M. Whiteaker J.R. L. N.L. S.J. D.C. A.G. P. Carr S.A. of the and of multiple reaction of proteins in Biotechnol. 2009; PubMed Scopus Google Scholar). the assay has the to large-scale verification of the hundreds of candidate biomarkers in a to clinical multiple reaction monitoring stable isotope standards and capture by antipeptide antibodies control. The of to candidate biomarkers in plasma is by the of quantitation of the assays. enrichment of the target MRM-MS is to measure proteins in the concentration range from small of plasma L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell. Proteomics. 2006; 5: 573-588Abstract Full Text Full Text PDF PubMed Scopus (1081) Google Scholar), the of human of the high of a small of proteins that detection of proteins. quantification of candidate biomarkers at concentrations in plasma, an enrichment be the success of using H. Addona T. Burgess M. Kuhn E. Carr S.A. assays for proteins in plasma by targeted mass spectrometry and stable isotope dilution.Mol. Cell. Proteomics. 6: Full Text Full Text PDF PubMed Scopus Google or enrichment Aebersold R. Zhang H. of from Chem. PubMed Scopus Google to targeted enrichment can be using antipeptide antibodies in SISCAPA assays isotope standards and capture by antipeptide N.L. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by antibodies Proteome Res. 2004; 3: PubMed Scopus Google Scholar). SISCAPA to it is feasible to measure candidate protein biomarkers in plasma at concentrations of using plasma and to the range by the capture J.R. L. L. A.G. and for high peptide enrichment and multiple reaction monitoring mass quantification of protein Cell. Proteomics. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). SISCAPA has been in a of platforms and N.L. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by antibodies Proteome Res. 2004; 3: PubMed Scopus Google Scholar, J.R. L. L. A.G. and for high peptide enrichment and multiple reaction monitoring mass quantification of protein Cell. Proteomics. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, with for quantitative analysis of protein 4: Google Scholar, A.N. Quantification of a serum by peptide enrichment and tandem mass spectrometry.Clin. Chem. 2008; PubMed Scopus Google Scholar, E. Addona T. Keshishian H. Burgess M. Mani D.R. Sabatine M.S. Gerszten R.E. Carr S.A. assays for and in plasma by peptide enrichment and targeted mass spectrometry.Clin. Chem. 2009; PubMed Scopus Google Scholar, H. J. peptide enrichment and liquid mass for total Chem. 2010; 56: PubMed Scopus Google Scholar, J.R. L. Zhang L.C. L. A.G. enrichment of peptides on for quantification of serum Biochem. PubMed Scopus Google Scholar). to SISCAPA has been in small at working assays for a of These the of the to multiplex and the to assays the of is from these is an of the with respect to the success and strategies for antibody the to assays on a and the of the to hundreds of target is for the feasibility of generating SISCAPA assays on a larger for in generating assays to hundreds of biomarkers or for such as in a human protein detection and quantitation N.L. C.H. A.G. Patterson Gillette M. Aebersold R. Carr S.A. A human proteome detection and quantitation Cell. Proteomics. 2009; 8: Full Text Full Text PDF PubMed Scopus Google Scholar). we these in the SISCAPA to by generating de novo a of individual SISCAPA assays and their describe a screening for working the success rate of the assay and the for of SISCAPA assays. human plasma plasma, was from the of and and at and were from used for of plasma was from antibodies were by Selected tryptic peptide with a at the were to a protein and used as immunogens for were and the the antibody on was chosen as the of antibodies were affinity from of using with the The concentrations of antibodies were by assay. peptides were from and were as peptides with and of were The of the peptides was as measured by stable isotope labeled the or was labeled with or and labeled In labeled peptides were with or and labeled and were the stable isotope the to use was by and concentrations were by analysis at or analysis was using an with a to a mass The A of and in and B of and in of peptide were a 0.5 and at by the B B B 100% B B analysis of by by with included and were by using a peptide and to detection of of the to and were The were for of the peptide for the of and for were in a using B. 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D.C. for and targeted 2010; PubMed Scopus Google for MRM development. human plasma was and to of The was to for with of and concentration were and then was to a of The was at for then from the and to at for To the of was concentration and the was and in the for of was and the was into The was to with was to and the were at The the was to with of was to and the was further for at 0.5 of was to the The digested plasma was using on a The were with of and with of of digested plasma were to with and individual by of for the and The peptides were from the with the peptide were and for at of plasma were in for use in SISCAPA enrichment were in was to a with a of peptides and antipeptide antibody for in the The of the was to 100 with the and were to the the were to at A of Protein were with for and to A with a was used for The were to the of and for The were to the for a in The was in the for a total of the the was to the the were to the of with and were for The the peptides was with and at analysis by mass peptide was in the of by the antibodies of antibody with and the by liquid with a was used for liquid used were and of the were a for at with B. the was with a 100 with The was at and of a of B from B in the of the the was with B for at a rate of and the was at B at The was to a mass with a interface in the instrument included a of an of a of and for and were in a of the were by of peptide and and were from a of in the B. D.M. N. M. B. R. D.C. for and targeted 2010; PubMed Scopus Google Scholar). MRM were to using a of and a of 0.5 across a for accurate A of total per peptide were for and MRM on the were by were a of and a factor of were and from peptides were by the of the peptides and stable were generated from and in an for The of this study were to determine the success rate of generating antipeptide antibodies for SISCAPA and to to the of a cost-effective assay pipeline with a high success an of the proteotypic peptides from a protein were used to SISCAPA affinity antibodies with quantitative mass spectrometric and the assays were by a screening that we to many peptides from a single protein target can be used to immunize a and many antipeptide antibodies to be for that target to at assay is multiple immunogens be used in the to and is the of antibody per peptide and many SISCAPA assays that is the and the of assay across a of is to that assay and of of (i.e. of detection and were not the of this to the as a and identify of to assay proteins were selected from a of proteins as to based on a PubMed strategy M. N.L. A of candidate biomarkers for targeted Google Scholar). was to the of proteins to that be more to assay development. protein proteotypic peptides were for assay development. The peptides were selected to (12Lange V. Picotti P. Domon B. Aebersold R. Selected reaction monitoring for quantitative proteomics: A tutorial.Mol. Syst. Biol. 2008; 4: 222Crossref PubMed Scopus (1131) Google Scholar, N.L. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by antibodies Proteome Res. 2004; 3: PubMed Scopus Google Scholar). In the peptides were selected to be be between and and or in the of or as as peptides (e.g. and were and of for proteins were a of tryptic peptides available for and these were In to the suitable peptides were based on or as a protein was the proteins were to by and peptides high in the mass were from the protein was not a of peptides was by proteomic (e.g. for a protein of we an to the peptides Mani D.R. Carr S.A. of peptides for targeted protein assays by mass Biotechnol. 2009; PubMed Scopus Google Scholar). proteins than five proteotypic the was further to and the is to that used are based on protein and such as and are of use for the to antibodies to capture the of tryptic peptides for SISCAPA assays D.R. B cell of Sci. 2005; PubMed Scopus Google Scholar). a of peptides as in order to success for assay In this 403 unique peptides were selected for assay representing up to five proteotypic peptides per protein proteins we were to identify five or more peptides the with proteins a single The protein targets and their peptides used for assay are in To the of at assay per target multiple proteotypic peptides from a single protein were to and used for immunization of individual rabbits. immunization from to were and with the majority of proteins five immunogens. were per multiplex immunization and the with the antibody on was chosen as the of To or antipeptide antibodies the antibody were for the majority of the 89 protein targets the of proteins targeted in the strategy of using multiple immunogens and of the peptides for affinity of A total of 220 antibodies were affinity from of per target and the antibody from to with a of In to the generation of and stable peptides were for of SISCAPA assays. In SISCAPA antibodies and and stable peptides) were generated for 220 peptides representing 89 proteins of the peptides and antibodies is an of the assay was on an to to and to for sequence and MRM development. peptides from and from the Mass to a at the been to undergo the or W. of peptides by liquid Mass 2003; PubMed Scopus Google Scholar). This be A from the was not stable in for MRM measurement not These peptide targets were not included in the from the peptides that were used to a in B. D.M. N. M. B. R. D.C. for and targeted 2010; PubMed Scopus Google Scholar), a available for MRM the a of the on was for The peptide standards were by monitoring the selected in from these were used to the to the with given to with than the The was in the to allow for MRM and to peptides for and MRM are available in the analysis of antibodies of a of protein concentration by and assay and an of peptides are or peptides to the antibody as a result of of the affinity A.N. Quantification of a serum by peptide enrichment and tandem mass spectrometry.Clin. Chem. 2008; PubMed Scopus Google Scholar, E. Addona T. Keshishian H. Burgess M. Mani D.R. Sabatine M.S. Gerszten R.E. Carr S.A. assays for and in plasma by peptide enrichment and targeted mass spectrometry.Clin. Chem. 2009; PubMed Scopus Google Scholar). antipeptide antibodies were generated to measure proteins using methods of this of the high of MRM of peptide from the antibody the working concentration range of the assay and the detection to peptides are not an for the screening we which antibodies peptides be for with these antibody was for peptide by antibody with Protein and with The was by using the methods to the peptide for peptide detection for target are available in of the antibodies for the of a peptides detection for assays to the range of ng/ml. A has been to the peptides affinity T. B. N. in for peptide enrichment in Scholar). and assays were available for further screening and peptides was not a for screening the antibodies with respect to SISCAPA capture and assay in the target stable peptide was into an tryptic of human plasma at concentrations and the peptide was into the plasma at a concentration were chosen to determine at the of detection and to of The to fmol/μl or protein concentration and enrichment from The to fmol/μl or the range of to fmol/μl and protein concentrations in the range of the high to fmol/μl or protein concentrations the by the peptide for of the detection at of the peptide or were into of based on a total of multiplex were to the assays. 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The of successful assays is from the by a are for of the of peptide immunogens and the of affinity of protein targets for which A and B assays were of protein targets for which A assays were of antibodies that were affinity of immunogens used per of antibodies that were affinity of immunogens used per in a new affinity and are major of the of SISCAPA assays. peptide in of and can in assay To the in peptide and the of this to assay we the detection for of the target peptides in this study in the of affinity of stable peptides was by MRM-MS and were used to the the by of at the of peptide for SISCAPA assay we can a to of A assays for the peptides In were of peptides that in the SISCAPA that a antibody can The is the large-scale study to the success rate of generating antipeptide antibodies for SISCAPA assays. the success rate for sensitive SISCAPA assays in this study was high this success rate is to that proteins for (e.g. The success rate for a sensitive assay to a target protein was using the multiplexing immunization strategy of five immunogens and antibodies per The of the assays was to in peptide enrichment J.R. L. L. A.G. and for high peptide enrichment and multiple reaction monitoring mass quantification of protein Cell. Proteomics. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, A.N. Quantification of a serum by peptide enrichment and tandem mass spectrometry.Clin. Chem. 2008; PubMed Scopus Google Scholar, E. Addona T. Keshishian H. Burgess M. Mani D.R. Sabatine M.S. Gerszten R.E. Carr S.A. assays for and in plasma by peptide enrichment and targeted mass spectrometry.Clin. Chem. 2009; PubMed Scopus Google Scholar). with such as enrichment using of plasma, SISCAPA at detection with less In we that SISCAPA assays can achieve in the range from larger of plasma J.R. L. L. A.G. and for high peptide enrichment and multiple reaction monitoring mass quantification of protein Cell. Proteomics. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). The in this study indicate that hundreds of SISCAPA assays can be generated per per of peptides of peptides and generation of antibodies and affinity of and of methods and the evaluation of This is for biomarker verification a of highly sensitive assays be in a time. a of the is by the of a of be the of a or more antibodies it is a small up to assays per this study that the of to larger (e.g. to a targets or be on the and of laboratories and antibody In these cost and cost-effective planning of generation of SISCAPA assays. a major cost of generating SISCAPA assays is the cost of the antipeptide for that this cost can be by immunization with at five peptide immunogens per cost associated with antibody generation is the affinity per results a high success rate for generating a of high assay per protein antibodies for to peptide immunogens from that target this study the range of of antibodies across a of immunogens. The was of antibody from of A single SISCAPA capture in the of SISCAPA can be given the antibody for hundreds to of assays. biomarker this of antibody is for an assay and verification on hundreds of patient the of generating a antibody for candidate proteins results in verification larger clinical be and antibodies to be it is to the from the used for antibody can be at a the been Methods for antipeptide antibodies that in SISCAPA assays been L. Whiteaker J.R. L.C. L. L. X. A.G. screening of antibodies for SISCAPA assays using a and liquid reaction spectrometry.J. 2010; PubMed Scopus Google Scholar, N.L. antibody of high affinity by a 2009; PubMed Scopus Google Scholar), and the to M. N.L. A for of antibodies for use in PubMed Scopus Google the large-scale production of a to be less be successful for generating high affinity antipeptide with

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