Global site-specific analysis of glycoprotein N-glycan processing

摘要

N-glycans contribute to the folding, stability and functions of the proteins they decorate. They are produced by transfer of the glycan precursor to the sequon Asn-X-Thr/Ser, followed by enzymatic trimming to a high mannose-type core and sequential addition of monosaccharides to generate complex-type and hybrid glycans. This process, mediated by the concerted action of multiple enzymes, produces a mixture of related glycoforms at each glycosite, making analysis of glycosylation difficult. To address this analytical challenge we developed a robust semi-quantitative MS-based method that determines the degree of glycan occupancy at each glycosite, and the proportion of N-glycans processed from high mannose type to complex type. It is applicable to virtually any glycoprotein, and a complete analysis can be conducted with 30 μg of protein or less. Here we provide a detailed description of the method that includes procedures for (1) proteolytic digestion of glycoprotein(s) with specific and non-specific proteases; (2) denaturation of proteases by heating; (3) sequential treatment of the glycopeptide mixture with two endoglycosidases, Endo H and PNGase F, to create unique mass signatures for the three glycosylation states; (4) LC-MS/MS analysis; (5) data analysis for identification and quantitation of peptides for the three glycosylation states. Full coverage of site-specific glycosylation of glycoproteins is achieved with up to thousands of high-confidence spectra hits for each glycosite. The protocol can be performed by an experienced technician or student/post-doc with basic skills for proteomics experiments and takes approximately 7 days to complete.