Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA

The ability to measure specific proteins of interest is critical to the basic sciences and clinical research. To this end, immunoaffinity-based assays such as Western blotting, immunohistochemistry, and ELISAs have been in use for decades, but have several shortcomings including difficulty in multiplexing, a lack of standardization, and a semi-quantitative nature (e.g. Western blotting and immunohistochemistry) (1). Recently, there has been tremendous growth in using the sensitive, specific, multiplexable, and quantitative technology, multiple reaction monitoring-mass spectrometry, to measure tryptic peptides as stoichiometric surrogates for the detection of proteins from complex samples (2–7). The sensitivity of targeted multiple reaction monitoring (MRM)1 is enhanced 103–104-fold by coupling it upstream with immunoaffinity enrichment of tryptic peptides in a peptide immuno-MRM assay (8–14). Advantages of immuno-MRM include high specificity, multiplexability (15, 16), and standardization, enabling high inter-laboratory reproducibility (17). The extent to which antibodies generated for immuno-MRM could support widely-used conventional immunoassay formats has not been investigated. This question is important because a lack of validated affinity reagents is a major obstacle to widespread implementation of immuno-MRM, which has considerable analytical advantages over traditional methods. Because the market for immuno-MRM is at present small relative to that for widely adopted conventional immunoassay formats (e.g. Western blotting and ELISA), commercial antibody suppliers are not incentivized to develop content specifically for immuno-MRM assays. Thus, we reasoned that if antibodies could be generated that are capable of supporting both conventional technologies as well as the emerging MRM platform, this might spark commercial interest by increasing the value of the antibodies, ultimately providing reagents to foster widespread implementation of immuno-MRM. Antigens used for antibody generation in conventional assays typically consist of either purified proteins, protein segments of 100–150 amino acids, or synthetic peptide sequences (18, 19). Antigenic prediction algorithms are often used to identify regions of target proteins that are most likely to be exposed on the surface of the protein and, thus, accessible for antibody binding. In contrast, proteotypic peptide antigens are selected for development of antibodies for immuno-MRM based on their uniqueness in the genome and their robust detectability by mass spectrometry, without regard to protein structure (because the protein will be proteolyzed during the assay). Because some widely used conventional immunoassay formats (e.g. Western blotting and indirect ELISA) detect proteins in their denatured form, it was reasonable to ask whether antibodies raised against short, linear, tryptic peptides would also work in these alternative formats. Here, we develop, characterize, and make publicly available 40 novel immuno-MRM assays and the associated monoclonals, and report the success rate of generating recombinant monoclonal antibodies (mAbs) that work in immuno-MRM assays. Furthermore, we determine the cross-over success rates of applying the mAbs in Western blotting and indirect ELISA assays.