FIGURE 3.

Representative nano-LC-MS/MS of PNGase F-deglycosylated tryptic-digested peptides of mammalian rLILRA3 confirmed four predicted N-glycosylation sites. A, in-gel peptide digestion of deglycosylated rLILRA3 with Glu-C showed deamidation of asparagine to aspartic acid at Asn¹⁴⁰ (panel i), Asn²⁸¹ (panel ii), and Asn⁴³¹ (panel iii) indicating N-linked glycosylation of these sites. B, digestion with chymotrypsin showed deamidation at Asn²⁸¹ (panel i) and Asn³⁴¹ (panel ii). C, digestion with trypsin detected Asn²⁸¹ (panel i) and Asn⁴³¹ (panel ii). It is noteworthy that some sites were detected in peptides digested by more than one enzyme, and none of the enzymes provided full peptide coverage. The predicted Asn³⁰² was not detected. The sequence of the peptide, the fragmentation pattern, and the detected fragment ions are shown at the top right in each panel. b ions contain the N-terminal region of the peptide; y ions contain the C-terminal region of the peptide. Deamidation of asparagine to aspartic acid is designated as “N” with an underscore.