Abstract
This manuscript describes the enrichment and mass spectrometric analysis of intact glycopeptides from mouse liver, which yielded site-specific N- and O-glycosylation data for ∼ 130 proteins. Incorporation of different sialic acid variants in both N- and O-linked glycans was observed, and the importance of using both collisional activation and electron transfer dissociation for glycopeptide analysis was illustrated. The N-glycan structures of predicted lysosomal, endoplasmic reticulum (ER), secreted and transmembrane proteins were compared. The data suggest that protein N-glycosylation differs depending on cellular location. The glycosylation patterns of several mouse liver and mouse brain glycopeptides were compared. Tissue-specific differences in glycosylation were observed between sites within the same protein: Some sites displayed a similar spectrum of glycan structures in both tissues, whereas for others no overlap was observed. We present comparative brain/liver glycosylation data on 50 N-glycosylation sites from 34 proteins and 13 O-glycosylation sites from seven proteins.
Highlights
The term protein glycosylation covers a wide variety of posttranslational modifications (PTMs)
A tryptic digest of mouse liver proteins was subjected to Lectin Weak Affinity Chromatography (LWAC) chromatography using wheat germ agglutinin
The sample yielded numerous targeted GlcNAcylated peptides, mainly from nuclear proteins, but the presence of other glycan oxonium ions indicated the additional enrichment of N- or O-glycopeptides in the secretory pathway
Summary
The term protein glycosylation covers a wide variety of posttranslational modifications (PTMs). Protein glycosylation studies have focused on the in-depth analysis of enzymatically or chemically released glycan pools (4 – 6). This approach is still the most reliable method for obtaining detailed structural information about the protein-modifying carbohydrates as the protein-level heterogeneity, both in terms of site occupancy and the number of site-specific structures, represent exceptional challenges for analysis. It should be noted that intact glycopeptide studies usually only allow the determination of glycan compositions; the identity of the oligosaccharide units and their linkage may be obtained from released glycan studies [5, 6] In this manuscript, we present data on the site-specific Nand O-glycosylation of mouse liver proteins. While individual proteins have been studied this way [15, 26], this is the first time that cellular-localization-specific and tissue-specific glycosylation have been compared on a larger scale at a glycosylation-site-specific level
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