Multidimensional Fractionation Is a Requirement for Quantitation of Golgi-Resident Glycosylation Enzymes from Cultured Human Cells Chi-Hung Lin, † Jenny H. L. Chik, † Nicolle H. Packer, † and Mark P. Molloy* ,†,‡ † Department of Chemistry and Biomolecular Sciences, Faculty of Science, and ‡ Australian Proteome Analysis Facility, Macquarie University, Sydney, Australia * S Supporting Information 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. KEYWORDS: Glycosylation, mass spectrometry, Golgi, proteomics, subcellular fractionation ■ INTRODUCTION Protein glycosylation is initiated and processed by a set of glycosylation enzymes in the secretory pathway. Biosynthesis of glycan chains is therefore regulated through modulation of the activities of these glycosylation enzymes. Gene expression serves as the primary control to provide the required enzyme activities for biosynthesis. Following gene expression, glyco- sylation enzymes are often subjected to fine-tuning through additional post-translational regulations. For example, homo- meric and heteromeric complexes of glycosylation enzymes are shown to promote the formation of specific glycan chains. 1,2 Cosmc, the core 1 O-glycan synthase (T synthase) specific chaperone, is required for functional T synthase to fold correctly and be transported to the Golgi apparatus. 3 Many glycosyltransferases (GTs) are also glycoproteins themselves, and often, glycosylation is important for correct folding and activity. 4 The fast growing field of glycomics provides analytical platforms to characterize glycans and measure alterations in their abundance during pathophysiological development and can lead to the identification of biomarkers and therapeutic targets. 5,6 Understanding how the synthesis of these disease associated glyco-epitopes is regulated would provide more insights into the functional roles of these glycans, which are mostly unknown. Upon detection of glycan epitopes that are differentially expressed, mRNA levels can be assessed to identify the expression of putative glycosylation enzymes that are linked to the oligosaccharide structural alterations. The finding that expression of functional T synthase is controlled post-transcriptionally by Cosmc suggests that it is important to also quantify the expression of the glycosylation enzymes directly. Information on the protein expression level of the glycosylation enzymes provides the link between the resultant glycan structure phenotype and the transcription of the genome, thereby allowing the understanding of the regulation of glycan biosynthesis in what could be described as a reverse- genomics approach. However, to date, robust proteomic approaches to detect and quantitate glycosylation enzymes are lacking. To quantify endogenous glycosylation enzymes is challeng- ing as most of these enzymes are Golgi resident membrane proteins and are usually present in low-molar abundance. Over the past decade, progress in Golgi membrane proteomics has led to the identification of a limited set of Golgi resident proteins. 7-10 In these studies, around 30 Golgi-resident GTs Received: July 28, 2014 Article pubs.acs.org/jpr © XXXX American Chemical Society A DOI: 10.1021/pr500785f J. Proteome Res. XXXX, XXX, XXX-XXX