hTRPA1 possesses four reactive Cys’s. (A, top) Treatment protocol for brief IA exposure of HEK293 cells expressing hTRPA1 for MS analysis. (bottom) Complete analysis of the percentage of each Cys adducted by IA, NEM, and NMM as determined by comparisons of identified MS/MS peaks. (B) Representative MS/MS spectra for tryptic peptide containing C621 (CAM modified) and C633 (NEM modified). (C) Representative MS/MS spectra for tryptic peptide containing C621 (CAM modified) and C633 (NMM modified). (B and C) Asterisks denote fragment ions with CAM modification. (D) Superimposed full scan mass spectra for the modified C621- and C633-containing tryptic peptide showing relative differences in peak intensity for each modified version (identified by different colors). Data are representative of the first analysis using a C18 column. Accurate mass-based (<5 ppm) reconstructed ion chromatograms were generated for each modified peptide, and the signal was averaged over the chromatographic peak width before superimposition. Overlay (3D) was performed in the Qual Browser (Thermo Fisher Scientific) data viewer followed by spectrum normalization to the largest peak in the scan with multiple scans normalized all the same. (E) Cartoon representation of hTRPA1 structure and approximate location of identified Cys’s (red indicates IA adduction). (F) Calculated rate reaction of IA for select hTRPA1 Cys’s, indicating the high reactivity of C621.