N- and O-Glycosylation in the Murine Synaptosome

Abstract

We present the first large scale study characterizing both N-and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N-and O-linked carbohydrate structures. Liquid chromatography-MS (LC/ MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N-and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues. Molecular & Cellular

O-Glycosylation of the N-terminal Region of the Serine-rich Adhesin Srr1 of Streptococcus agalactiae Explored by Mass Spectrometry

“…Determination of Glycosylation Sites-ETD has been shown to be a valuable tool for the analysis of glycopeptides because the glycosidic bond can be preserved during the fragmentation process, allowing glycosylation sites to be accurately mapped (10,67). Srr1 digest was therefore analyzed in both untargeted/targeted approaches using ETD as the fragmentation mode.…”

Carbamidomethylation Side Reactions May Lead to Glycan Misassignments in Glycopeptide Analysis

“…O-linked glycans may feature different core structures, among them there is a deoxyhexose, fucose (Fuc). We have reported that in a mouse synaptosome dataset numerous high scoring O-fucosyl peptides were detected (Table 1) [21].…”

“…Further details about the synaptosome preparation, and the fractionation of the digest have been published [21,28].…”

“…The activation of multiply charged glycopeptide ions by electron-transfer leads to peptide backbone cleavages leaving the sidechains intact in principal. This is an advantage and a significant drawback at the same time, since a wide array of glycoforms may be present in each mixture analyzed [21,24,25]. In order to interpret such results, all potential glycans have to be considered as variable modifications, and thus, not only a protein, but also a glycan database has to be interrogated.…”

Extracellular Protein Phosphorylation, the Neglected Side of the Modification

“…Luckily, such prediction-based information has been available for several proteins, and is regularly listed in UniProt. In a previous large scale glycosylation study, we found this information very reliable, we encountered only two examples among hundreds of assignments that indicated glycosylation on a predicted cytoplasmic domain (45). Also, there are several topology prediction servers available to assess potential localization of the modification sites, of which CCTOP was used here for topology prediction of the ER/ Golgi-resident phosphoproteins.…”