Proteomic identification of specific glycosyltransferases functionally implicated for the biosynthesis of a targeted glyco-epitope

Proteomics
Chi-Hung LinKay-Hooi Khoo

Abstract

Functional glycomic and glycoproteomic analyses often entail correlating the mapped glycosylation pattern of a cell against the activities of specific glycosyltransferases it expresses. While the mRNA transcripts can be readily mapped, the expression of a functional glycosyltransferase at protein level has defied most current proteomic approaches. To enable identification of these low abundant Golgi residing membrane bound proteins, we have developed a novel semigel-based shotgun proteomic workflow incorporating subcellular fractionation and one-step affinity enrichment of the detergent solubilized Golgi preparation on resins derivatized with nucleotide diphosphates. Applying the strategy to a colonic adenocarcinoma, Colo205, which is known to aberrantly synthesize abundant fucosylated extended type 1 chain, we first validated that beta3-galactosyltransferase 5 (beta3GalT5) is indeed the overexpressed beta3GalT. This and beta4GalT1 are the two galactosyltrasferases which were positively identified by proteomic analysis of the eluted fractions from uridine diphosphate (UDP)-affinity column. Substituting UDP with a guanidine diphosphate (GDP)-affinity column and monitoring the eluted fractions for enriched alpha3/4-fucosyltransfe...Continue Reading

References

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