Figure 5.

Quantitative peptidomics analysis scheme for the characterization of the substrate specificity of CPZ using a tryptic peptide library. (A) Schematic representation of the quantitative peptidomics analysis and (B–E) representative results. Tryptic peptides were prepared from the digestion of five selected proteins with trypsin. The resultant peptide library was aliquoted and incubated with no enzyme or different CPZ concentrations (i.e., 1, 10, and 100 nM) for 16 h at 37 °C. After incubation samples were labeled with one of four stable isotopic TMAB tags as follows: D0 for the sample without enzyme; D3 for 1 nM CPZ; D6 for 10 nM CPZ; and D9 for 100 nM CPZ. All the individual reactions were pooled and analyzed by LC-MS. Representative examples are shown for (B) non-substrates, (C) good substrates, (D) weak substrates, and (E) products of CPZ.