Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains
(A) Quantitative peptidomics scheme. Tryptic peptides were obtained from digestion of five selected proteins (BSA, bovine thyroglobulin, bovine α-lactalbumin and human α and β–hemoglobin) with trypsin. The resultant peptide library was aliquoted and digested with no enzyme or different rhCPD concentrations of 0.1, 1, 10, and 100 nM for 16 h at 37°C. After incubation samples were labeled with one of five stable isotopic TMAB-NHS tags (D0 = 100 nM; D3 = 10 nM; D6 = 1 nM; D9 = 0.1 nM; D12 = No enzyme). Then, samples were pooled and analyzed by LC-MS. Examples of representative data are shown for (B) non-substrates, (C) good substrates, (D) weak substrates and (E) products.