Protein glycosylation is synthesized through concerted actions of glycosylation enzymes in the secretory pathway. Evidences have shown that functions of these enzymes are not only regulated at gene expression level but also post-transcriptionally at protein level. We have previously developed MRM_HR assays to quantitate differential expressions of endogenous glycosyltransferases. However, robust enzyme activity assays to detect endogenous glycosyltransferase activities is lacking in our proposed systems biology approach to study glycan biosynthesis.

Lectins are glycan binding proteins that could bind and distinguish structural isomers. We previously developed MRM_HR assays to analyze and quantitate degree of sialylation of glycopeptides from glycoproteins. These properties suggest lectin and MRM_HR analysis are useful tools to detect change of glycan structures before and after enzyme reaction.

In this study, we developed a novel glycosyltransferase assay that combines lectin detection and MRM_HR analysis. We demonstrated our method by detecting α2-6 sialyltransferase activity. We tested binding specificities of three sialic acid binding lectins, SNA, MALI and MALII using glycosidase modified fetuin and confirmed SNA binds specifically to α2-6 sialic acid. De-sialylated fetuin was immobilized on 96-well plate as substrate for sialyltransferase reaction. Probing by SNA showed only reaction with recombinant ST6Gal1 gave positive signal suggesting addition of sialic acid in α2-6 linkage on substrate. Trypsin digestion was then conducted in the same wells and peptide mixtures were analyzed by MRM_HR assays. The results confirmed the addition of sialic acid on fetuin and degree of sialylation could be calculated accordingly.

Our results showed this method is superior in that it is robust and compatible with cell lysate so that it could be used for high throughput screening. MRM_HR analysis provided quantitative results and confirmed the signal from SNA detection is truly from immobilized substrate. Thus, the method could be configured to fit different analysis requirements.