title: Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.
abstract: Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Faculty of Science, and ‡Australian Proteome Analysis Facility, Macquarie University , Sydney, Australia.
authors.forename: Chi-Hung
authors.initials: CH
authors.lastname: Lin
authors.forename: Jenny H L
authors.initials: JH
authors.lastname: Chik
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
authors.forename: Mark P
authors.initials: MP
authors.lastname: Molloy
mesh_terms: D002240:Carbohydrate Sequence; D045744:Cell Line, Tumor; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D004798:Enzymes; D011134:Polysaccharides
keywords: Glycosylation; Golgi; mass spectrometry; proteomics; subcellular fractionation