Dataset Information

Tissue-Specific Glycosylation at the Glycopeptide Level.

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.

SUBMITTER: Medzihradszky KF

PROVIDER: S-EPMC4528240 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Tissue-Specific Glycosylation at the Glycopeptide Level.

Medzihradszky Katalin F KF Kaasik Krista K Chalkley Robert J RJ

Molecular & cellular proteomics : MCP 20150520 8

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), secre ...[more]

PMID: 25995273

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Project description:Glycosylation is a common protein modification with a significant role in many vital cellular processes and human diseases, making the characterization of protein-attached glycan structures important for understanding cell biology and disease processes. Direct analysis of protein N-glycosylation by tandem mass spectrometry of glycopeptides promises site-specific elucidation of N-glycan microheterogeneity, something that detached N-glycan and deglycosylated peptide analyses cannot provide. However, successful implementation of direct N-glycopeptide analysis by tandem mass spectrometry remains a challenge. In this work, we consider algorithmic techniques for the analysis of LC-MS/MS data acquired from glycopeptide-enriched fractions of enzymatic digests of purified proteins. We implement a computational strategy that takes advantage of the properties of CID fragmentation spectra of N-glycopeptides, matching the MS/MS spectra to peptide-glycan pairs from protein sequences and glycan structure databases. Significantly, we also propose a novel false discovery rate estimation technique to estimate and manage the number of false identifications. We use a human glycoprotein standard, haptoglobin, digested with trypsin and GluC, enriched for glycopeptides using HILIC chromatography, and analyzed by LC-MS/MS to demonstrate our algorithmic strategy and evaluate its performance. Our software, GlycoPeptideSearch (GPS), assigned glycopeptide identifications to 246 of the spectra at a false discovery rate of 5.58%, identifying 42 distinct haptoglobin peptide-glycan pairs at each of the four haptoglobin N-linked glycosylation sites. We further demonstrate the effectiveness of this approach by analyzing plasma-derived haptoglobin, identifying 136 N-linked glycopeptide spectra at a false discovery rate of 0.4%, representing 15 distinct glycopeptides on at least three of the four N-linked glycosylation sites. The software, GlycoPeptideSearch, is available for download from http://edwardslab.bmcb.georgetown.edu/GPS .

Dataset Information

Tissue-Specific Glycosylation at the Glycopeptide Level.

Publications

Tissue-Specific Glycosylation at the Glycopeptide Level.

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