In vivo and in vitro MS-based peptide profiling experiments. (a) In vivo global peptide profiling reveals that the DPP4 cleavage product Mepβ(25-41) is elevated in the DPP4^+/+ sample, while higher levels of the DPP4 substrate, Mepβ(21-41), are detected in the DPP4^−/− samples. (b) In vitro incubation of the Mepβ(25-41) peptide with kidney brush border membranes from DPP4^+/+ and DPP4^−/− results in a series of aminopeptidase cleavage products. (c) In contrast, incubation of Mepβ(21-41), a proline containing DPP4 substrate, with DPP4^+/+ membranes results in dipeptide cleavage products that are not produced with DPP4^−/− membranes. The data also indicates a lack of aminopeptidase activity adjacent to penultimate proline containing peptides. (d) Brush border membrane experiments with the model peptide substrates 3P and 5P show that brush border membrane peptidase activity is able produce DPP4 substrates. (e) Incubation of 3P and 5P with brush border membranes from APN/A^+/+ and APN/A^−/− mice indicate a direct role for brush border membrane AP activity in the production of DPP4 substrates. (f) A model showing the interlinked relationship between AP and DPP4 activities during the degradation of proline-containing peptides. For all graphs, data represent mean values ± s.e. (*, p <0.05; **, p<0.01), Student's t-test.