Immunoproteomics Using Polyclonal Antibodies and Stable Isotope–labeled Affinity-purified Recombinant Proteins

Abstract

The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope–labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A–coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope–labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed. The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope–labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A–coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope–labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed. Mass spectrometry–based proteomics is fast developing in the direction of clinical applications. Therefore, reliable quantification methods for absolute protein concentration determination are indispensible tools for future applications. So far, enzyme-linked immunosorbent assays and similar antibody-based methods excel in the sensitive detection of low levels of proteins in complex matrices, whereas mass spectrometry enables unbiased approaches and can provide unsurpassed specificity. The fact that most proteomes have a very high dynamic range between high and low abundant proteins, in particular for clinical samples, such as plasma and serum, often makes it necessary to use protein depletion of the most abundant proteins (1.Bjorhall K. Miliotis T. Davidsson P. Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples.Proteomics. 2005; 5: 307-317Crossref PubMed Scopus (325) Google Scholar, 2.Steel L.F. Trotter M.G. Nakajima P.B. Mattu T.S. Gonye G. Block T. Efficient and specific removal of albumin from human serum samples.Mol. Cell. Proteomics. 2003; 2: 262-270Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) and/or elaborate fractionations (3.Jin W.H. Dai J. Li S.J. Xia Q.C. Zou H.F. Zeng R. Human plasma proteome analysis by multidimensional chromatography prefractionation and linear ion trap mass spectrometry identification.J. Proteome Res. 2005; 4: 613-619Crossref PubMed Scopus (78) Google Scholar, 4.Pieper R. Gatlin C.L. Makusky A.J. Russo P.S. Schatz C.R. Miller S.S. Su Q. McGrath A.M. Estock M.A. Parmar P.P. Zhao M. Huang S.T. Zhou J. Wang F. Esquer-Blasco R. Anderson N.L. Taylor J. Steiner S. The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins.Proteomics. 2003; 3: 1345-1364Crossref PubMed Scopus (466) Google Scholar, 5.Pernemalm M. Lewensohn R. Lehtio J. Affinity prefractionation for MS-based plasma proteomics.Proteomics. 2009; 9: 1420-1427Crossref PubMed Scopus (81) Google Scholar) before running the mass spectrometry analysis. This has prompted several investigators to introduce a protein or peptide capture step using specific antibodies to allow for immunoaffinity enrichment prior to the MS analysis. In this way, a “sandwich” assay is obtained, but instead of having a readout in the analysis step based on a second antibody, the analysis step is performed using MS. In such an approach, either the intact protein is captured using an anti-protein antibody M. S. P. of in serum using and a of PubMed Scopus Google Scholar) or a peptide from the protein is captured using an antibody that has to the target peptide of N.L. Anderson Mass of peptides and proteins using and by Proteome Res. 3: PubMed Scopus Google Scholar, Zhao M. M. Anderson N.L. of quantitative proteomic assay using peptide mass Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar, J. peptide immunoaffinity enrichment and assay for PubMed Scopus Google Scholar, of a serum by immunoaffinity peptide enrichment and mass PubMed Scopus Google Scholar, analysis of an of from serum by and mass Proteome Res. 2009; PubMed Scopus Google This is the stable standards and capture by antibodies used stable standards and capture by protein albumin stable by in cell used stable standards and capture by protein albumin stable by in cell developed by Anderson and M. Miller Anderson N.L. of peptides from human plasma by mass Proteome Res. PubMed Scopus Google Scholar, N.L. peptide enrichment on magnetic using an trap Cell. Proteomics. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, Zhao Anderson enrichment of peptides on magnetic for quantification of serum PubMed Scopus Google Scholar, Zhao Anderson and method for high peptide immunoaffinity enrichment and multiple mass quantification of protein Cell. 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