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

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 beta 3-galactosyltransferase 5 (beta 3GalT5) is indeed the overexpressed beta 3GalT. This and beta 4GalT1 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 alpha 3/4-fucosyltransferase (FucT) activities, we then identified FucT3 and FucT6 as the two major alpha 3/4FucTs expressed in Colo205 at the protein level. Our proteomic analysis demonstrated that not all GDP-utilizing glycosyltransferases bind and are retained similarly by the GDP-affinity column and that specific activity assay along with optimization of binding and elution conditions is critical for successful identification of a particular subset of the targeted glycosyltransferases. Only OFUT1, a protein O-FucT, was additionally identified to coelute with the alpha 3/4FucT activity, and not other GDP-fucose utilizing FucTs.