Mirror-Cutting-Based Digestion Strategy Enables the In-Depth and Accuracy Characterization of N-Linked Protein Glycosylation.

Abstract

N-linked glycosylation plays important roles in multiple physiological and pathological processes, while the analysis coverage is still limited due to the insufficient digestion of glycoproteins, as well as incomplete ion fragments for intact glycopeptide determination. Herein, a mirror-cutting-based digestion strategy was proposed by combining two orthogonal proteases of LysargiNase and trypsin to characterize the macro- and micro-heterogeneity of protein glycosylation. Using the above two proteases, the b- or y-ion series of peptide sequences were, respectively, enhanced in MS/MS, generating the complementary spectra for peptide sequence identification. More than 27% (489/1778) of the site-specific glycoforms identified by LysargiNase digestion were not covered by trypsin digestion, suggesting the elevated coverage of protein sequences and site-specific glycoforms by the mirror-cutting method. Totally, 10,935 site-specific glycoforms were identified from mouse brain tissues in the 18 h MS analysis, which significantly enhanced the coverage of protein glycosylation. Intriguingly, 27 mannose-6-phosphate (M6P) glycoforms were determined with core fucosylation, and 23 of them were found with the "Y-HexNAc-Fuc" ions after manual checking. This is hitherto the first report of M6P and fucosylation co-modifications of glycopeptides, in which the mechanism and function still needs further exploration. The mirror-cutting digestion strategy also has great application potential in the exploration of missing glycoproteins from other complex samples to provide rich resources for glycobiology research.