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. 2017 Apr 20;8(31):51296–51316. doi: 10.18632/oncotarget.17261

Figure 7. PEITC targets the catalystic cysteine in UCH37.

Figure 7

(A) rUCH37/ADRM1 complex (1:1.3 molar ratio) was incubated with 1.5 mM PEITC for 15 min at 25°C and then treated with Biotin-Ub-PA/VME (an equal mix of both probes) for 5 min at 25°C. Coomassie stain of gel depicted. (B) Strategy for identifying PEITC labeled UCH37 cysteines. Samples (+/- PEITC) are initially treated with deuterated N-ethylmaleimide (d5-NEM), then denatured in the presence of dithiothreitol (DTT) to reverse the PEITC modification. Samples are then treated with unlabeled d0-NEM to label newly exposed cysteines and subjected to trypsin digestion. The resulting peptide samples are analyzed by LC-MS/MS. Peptides containing cysteine residues that were modified by PEITC will have a higher ratio of d0-NEM/d5-NEM labeled peptides compared to cysteine residues that did not form a PEITC adduct. (C) Schematic of the tryptic peptide containing the UCH37 active site cysteine residue. The active site cysteine (Cys88) is highlighted in red, while the second non-active site cysteine (Cys100) is highlighted in blue. The y17 and y27 ions fragmentation sites are also indicated. (D) MS1 and MS2 analysis of the C88/C100 tryptic peptide from the PEITC treated sample. Averaged d0/d0, d0/d5, and d5/d5 isotopic envelopes are displayed as well as the y17 and y27 fragmentation ions for the d0/d5 parent ion. (E) MS1 and MS2 analysis of the Cys88/Cys100 tryptic peptide from the untreated sample. Averaged d0/d0, d0/d5, and d5/d5 isotopic envelopes are displayed as well as the y17 and y27 fragmentation ions for the d0/d5 parent ion.