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

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.

Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots

“…In a recent study, leptin was also not detected in human plasma using 1D LC/MRM-MS; however, detection was possible with the increased separation power of 2D LC/ MRM-MS (47). Alternatively, immunoaffinity enrichment prior to LC/MRM-MS can also provide access to proteins in low ng/ml to high pg/ml range in human plasma (59,60). Both 2D separations and immunoaffinity enrichment will be investigated in future experiments in attempt to expand the number of proteins quantified from DBS samples.…”

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry–Based Assays

“…Serendipitously, these amino acids are among the highest frequencies found in nature, and will be present in most peptides. There are some caveats to this list: (1) hydrophobic amino acids such as Ile, Leu and Val can be problematic due to their slow hydrolysis rate, (2) in the presence of phenol (e.g., 0.2%), Tyr is stable during acid hydrolysis and plays an important role in peptide quantification by UV spectroscopy, and (3) the basic amino acids Arg, His, and Lys have longer retention times on reversed phase-ion exchange HPLC columns compared with other amino acids. Under typical acid hydrolysis conditions, Trp is readily destroyed by oxidation.…”

“…Towards these goals, we have: (i) coordinated a consensus approach to outline recommendations for the development of different classes of targeted mass spectrometry (MS)-based assays using a fit-for-purpose approach (2), (ii) launched the CPTAC Antibody Portal (3) (antibodies.cancer.gov) to facilitate the production, characterization and distribution of renewable affinity reagents to the community in order to support protein/peptide measurement and analysis, and (iii) launched and begun to populate the CPTAC Assay portal (4) (https://assays.cancer.gov/) to disseminate highly characterized targeted MS-based assays to the community, via access to standard operating protocols (SOP), reagents, and assay characterization data.…”

Applications of targeted proteomics in systems biology and translational medicine

“…Other immune‐MRM efforts are conducted by the Paulovich laboratory to overcome limit of detection issues in complex samples and eliminate the current bottleneck of translating biomarkers found in basic science studies to clinical practice 114. Further advances in the field concern the generation of immuno‐MRM monoclonal antibodies suitable for SRM and conventional antibody applications 115.…”