Amino-blocking sialic acid linkage specific alkylamidation for qualitative and quantitative analysis of sialylated glycoproteins.

Protein glycosylation is a pivotal post-translational modification involved in diverse physiological processes. Sialic acid, a terminal monosaccharide in glycans, exhibits critical biological functions and disease associations, yet its linkage isomers (α2,3- vs. α2,6-) remain challenging to be discriminated by conventional LC-MS/MS due to identical masses and similar chromatographic behaviors. Although sialic acid linkage-specific alkylamidation (SALSA) based on lactone ring-opening ammonolysis has been widely applied to glycopeptides, we observed undesired glycopeptide-glycopeptide condensation side reactions during derivatization. To overcome this limitation, we developed aSALSA, a strategy that incorporates amino-blocking of glycopeptides via reductive diethylation using acetaldehyde or its isotopically labeled counterpart (acetaldehyde-13C2) prior to SALSA derivatization, where side reaction prevention and quantitative isotopic labelling are achieved simultaneously. The optimal aSALSA protocol was achieved with reactions of two standard glycopeptides and one standard glycoprotein. By combining stable isotopic diethyl labeling with aSALSA, we quantitatively compared serum N-glycoproteomes from syphilis patients and healthy controls, and 106 differentially expressed intact N-glycopeptides were identified including α2,3- and α2,6- linkage isomers. aSALSA perfectly realizes simultaneous prevention of side reaction and quantitative isotopic labelling in linkage-specific derivatization of sialylated glycopeptides, and thus may find wide application in quantitative linkage-specific analysis of sialylated glycoproteins.