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. 2015 Feb;14(2):382-98.
doi: 10.1074/mcp.O114.043133. Epub 2014 Dec 15.

Anti-peptide monoclonal antibodies generated for immuno-multiple reaction monitoring-mass spectrometry assays have a high probability of supporting Western blot and ELISA

Regine M Schoenherr  1 , Richard G Saul  2 , Jeffrey R Whiteaker  1 , Ping Yan  1 , Gordon R Whiteley  2 , Amanda G Paulovich  3
Affiliations

Anti-peptide monoclonal antibodies generated for immuno-multiple reaction monitoring-mass spectrometry assays have a high probability of supporting Western blot and ELISA

Regine M Schoenherr et al. Mol Cell Proteomics. 2015 Feb.

Abstract

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach.

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Figures

Fig. 1.
Fig. 1.
Characterization of assays and antibody reagents. Immuno-MRM-MS response curve data in linear A, and log space B, and Western blot C, and ELISA D, data for the monoclonal antibody to peptide target GLQTSQDAR from the protein calreticulin (CALR; target peptide ID SAIC-16D). For the response curves, the heavy-to-light peak area ratios for the peptide's transitions are plotted versus the heavy peptide's theoretical concentration; y5 refers to the product ion; SUM refers to the response curve for which the peak areas of all transitions were summed before taking the peak area ratios; the error bars indicate the minimum and maximum (i.e. range) of the peak area ratios of the three capture and LC-MRM-MS replicates. For the Western blot, the expected molecular weight of calreticulin is 48 kDa (UniProt). For the ELISA assay, the B50% value was used as a relative value to assess the different mAbs' performances in these experiments; it indicates the inflection point of the curve and represents the mAb concentration at half of the calculated maximum binding of the mAb.
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