Edfors F, et al. (2014) “Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins.” Mol Cell Proteomics. 13(6): 1611-24. doi: 10.1074/mcp.M113.034140
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, 2) and/or elaborate fractionations (3⇓–5) 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 (6) or a peptide derived from the protein is captured using an anti-peptide antibody that has been raised to the target peptide of interest (7⇓⇓⇓–11). This is the principle behind stable isotope standards and capture by anti-peptide antibodies (SISCAPA),1 developed by Anderson and co-workers (12⇓⇓–15). In immunoaffinity proteomics, it is preferable for the affinity of the anti-peptide capture antibody to be high, but the requirement for high selectivity is lower, because the mass spectrometer can readily distinguish and quantify the analyte peptide of interest despite the binding of other peptides in the digested sample.
A disadvantage with the immunoaffinity proteomics strategy is the limited availability of suitable antibodies that recognize peptides from the corresponding protein targets. The affinity enrichment of peptides usually requires the generation of custom antibodies for each target peptide, and this very time-consuming process makes high-throughput efforts very difficult to pursue. Most efforts so far have been aimed toward generating monoclonal antibodies against specific peptides selected as appropriate for mass spectrometric detection, which is a laborious and costly exercise. It would therefore be of great interest to explore whether antibodies generated toward native proteins or protein fragments could be used for the capture of peptides and in this way take advantage of the huge resource of already existing reagents for immunoproteomics.
Here, we investigated whether the publicly available resources on polyclonal antibodies could be used for immuno-enrichment followed by quantitative proteomics. According to the Antibodypedia portal, there exist more than a million publicly available antibodies toward human protein targets, and more than 70% of these antibodies are polyclonal antibodies. These antibodies are of course interesting starting points as a resource for immunoproteomics, although this application was not intended at the time when the antibodies were generated. More specifically, we have investigated the use of polyclonal antibodies from the Human Protein Atlas project, covering more than 80% of all human protein-coding genes. These antibodies have been raised against human recombinant proteins called protein epitope signature tags (PrESTs), and we have therefore investigated the direct use of this resource for quantitative proteomics.
An attractive strategy for quantitative proteomics using immuno-enrichment is to use stable isotope approaches, including methods based on adding stable isotope–labeled peptides (16, 17), proteins (18, 19), or protein fragments (20). These methods are built on the detection of peptides generated by protease cleavage of the proteins in the sample, and the quantification is achieved by reading out the ratio between the endogenous peptide and the heavy-labeled spiked-in peptide. Because the endogenous protein and the labeled internal standard behave identically throughout the sample preparation including the immuno-enrichment, the relative ratio provides quantitative information, as the peptides can be distinguished by the mass spectrometer because of the shift in mass. We recently described (20) a method for protein quantification making use of the large library of PrESTs that has been developed in the course of the Human Protein Atlas (21) project. Heavy isotope–labeled PrESTs were quantified against an ultrapurified and accurately quantified protein standard using the albumin binding protein (ABP) tag. Thereafter, known amounts of heavy PrESTs were spiked into cell lysates, and the SILAC ratios were used to determine the cellular quantities of the endogenous proteins. That approach sidesteps the quantification-, storage-, and digestion-related causes of quantification error that are inherent to peptide-based methods. The PrEST-SILAC principle was used to simultaneously determine the copy numbers of 40 proteins in HeLa cells demonstrating quantitative measurements over a wide range of protein abundances, from the highly abundant cytoskeletal protein Vimentin, with 20 million copies, down to the low abundant transcription factor FOS, with only 6000 copies per cell.
Here, we combined the use of polyclonal antibodies for immunocapture with quantitative proteomics using heavy isotope–labeled proteins. A semi-automated immuno-SILAC method was developed for multiplex analysis of protein targets, taking advantage of the linear epitopes of the antibodies. A special effort was made to decrease the amounts of antibodies used in the assay. Based on the results, a new strategy for rapid mass spectrometry readout for target-specific proteomics is outlined in which antibodies are used for the multiplex immunocapture of peptides generated via trypsin digestion of cell extracts spiked with isotope-labeled recombinant protein fragments corresponding to the protein targets.