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

Fredrik Edfors,Tove Boström,Björn Forsström,Marlis Zeiler,Henrik Johansson,Emma Lundberg,Sophia Hober,Janne Lehtiö,Matthias Mann,Mathias Uhlen

doi:10.1074/mcp.m113.034140

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.

Highlights

Combinant 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
These antibodies have been raised against human recombinant proteins called protein epitope signature tags (PrESTs), and we have investigated the direct use of this resource for quantitative proteomics
The method relies on the use of stable isotope–labeled recombinant protein fragment standards (PrESTs) produced in bacteria (Fig. 1A)

Summary

Technological Innovation and Resources

Immunoproteomics Using Polyclonal Antibodies and Stable Isotope–labeled Affinity-purified Recombinant Proteins*□S. 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. 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. 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

EXPERIMENTAL PROCEDURES

RESULTS

Number of peptides

DISCUSSION