Figure 5

Limited proteolysis of the recombinant α-DG mucin-like domain, α-DG(316–484). Limited proteolysis of the α-DG(316–484) peptide (~5-10 μM) was carried out with 20 nM trypsin in PBS, pH 7.4 at room temperature. Aliquots of the reaction mix were collected at increasing times and analyzed by SDS-PAGE. Before incubation with trypsin (t 0), α-DG(316–484) already displays two bands corresponding to the entire peptide, α-DG(316–484), and to the slightly smaller truncated peptide α-DG(316–461), characterized by CE-ESI MS and originated via a C-terminal breakdown event. Noteworthy, the electrophoretic mobility of the whole α-DG(316–484) is much lower than expected on the basis of its molecular mass (18252 Da). Trypsin digests both the peptides producing one lower band at about 18 kDa (arrow), which is further fragmented, and completely degrades them in about 1 h.