1D LC-MS/MS proteomic profiling of MCF10A, HeLa, Hep G2, and HEK293 cells using peptide-based immunoaffinity enrichment and 105 commercially available monoclonal antibodies to proteins associated with the DNA damage response (DDR) network
Immunoaffinity enrichment of peptides coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM) enables highly specific, sensitive, and precise quantification of peptides and post-translational modifications. Major obstacles to developing a large number of immuno-MRM assays are the poor availability of monoclonal antibodies (mAbs) validated for immunoaffinity enrichment of peptides and the cost and lead time of developing the antibodies de novo. Although many thousands of mAbs are commercially offered, few have been tested for application to immunoaffinity enrichment of peptides. In this study we tested the success rate of using commercially available mAbs for peptide immuno-MRM assays. We selected 105 commercial mAbs (76 targeting non-modified “pan” epitopes, 29 targeting phosphorylation) to proteins associated with the DNA damage response network. We found that 8 of the 76 pan (11%) and 5 of the 29 phospho-specific mAbs (17%) captured tryptic peptides (detected by LC-MS/MS) of their protein targets from human cell lysates. Seven of these mAbs were successfully used to configure and analytically characterize immuno-MRM assays. By applying selection criteria, the results indicate a success rate up to 24% is possible, establishing the feasibility of screening a large number of catalog antibodies to provide readily-available assay reagents.
Sample Processing Protocol
MCF10A, HeLa, Hep G2, and HEK293 cell lines were used untreated, or after having been treated with irradiation (10 Gy, harvested after 2 hours after irradiation). Whole cell lysates were digested using trypsin, and peptides were enriched by immunoaffinity using the 76 pan antibodies for untreated cell lysate digests, and the 29 phosphorylation antibodies for the treated cells. To this end, the mAbs were multiplexed into groups of 2-13 mAbs depending on confirmed expression data available for the commercial mAbs. Each immunoaffinity enrichment experiment used 1 mg of a cell line’s digest, 1 ug of each mAb, and 1.5 uL of Protein G magnetic beads, and after overnight incubation the peptides were eluted off the antibodies using acid. Each capture with a particular mAb panel and digested lysate was performed in singlicate, and each eluate was analyzed by a shotgun MS/MS and a pseudo-targeted AIMS run (Accurate Inclusion Mass Screening). The LC-MS/MS analyses were performed on a nanoAcquity HPLC (Waters) coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific).
Data Processing Protocol
The raw MS/MS spectra were searched against version 3.69 of the Human International Protein Index (IPI) sequence database using MaxQuant/Andromeda, with the tryptic enzyme constraint set for up to two missed cleavages, oxidized methionine and phosphorylated serine, threonine, and tyrosine set as variable modifications, carbamidomethylated cysteine set as a static modification, and peptide MH+ mass tolerances set at 20 ppm. The overall FDR was set at ≤1% based on a decoy database search.