Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove N-glycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the N-glycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.
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PMID:Site-specific glycan-peptide analysis for determination of N-glycoproteome heterogeneity. 2409 84

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein's central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 N-glycan consensus sites were identified as glycosylated. Fifty-six N-glycopeptides covering 10 N-glycan sites, and 14 O-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins.
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PMID:Multiple enzyme approach for the characterization of glycan modifications on the C-terminus of the intestinal MUC2mucin. 2540 38

SugarQb (www.imba.oeaw.ac.at/sugarqb) is a freely available collection of computational tools for the automated identification of intact glycopeptides from high-resolution HCD MS/MS datasets in the Proteome Discoverer environment. We report the migration of SugarQb to the latest and free version of Proteome Discoverer 2.1, and apply it to the analysis of PNGase F-resistant N-glycopeptides from mouse embryonic stem cells. The analysis of intact glycopeptides highlights unexpected technical limitations to PNGase F-dependent glycoproteomic workflows at the proteome level, and warrants a critical reinterpretation of seminal datasets in the context of N-glycosylation-site prediction.
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PMID:Analysis of PNGase F-Resistant N-Glycopeptides Using SugarQb for Proteome Discoverer 2.1 Reveals Cryptic Substrate Specificities. 2977 40

Cell-surface N-glycans play important roles in both inter- and intracellular processes, including cell adhesion and development, cell recognition, as well as cancer development and metastasis; detailed structural characterization of these N-glycans is thus paramount. Here we report our comparative N-glycomics study of cell-surface N-glycans of the hepatocellular carcinoma (HCC) HepG2 cells vs the normal liver LO2 cells. With sequential trypsin digestion of proteins, C18 depletion of peptides without glycosylation, PNGase F digestion of N-glycopeptides, PGC enrichment of N-glycans, CH3I permethylation of the enriched N-glycans, cell-surface N-glycomes of the HepG2 and LO2 cells were analyzed using C18-RPLC-MS/MS (HCD). With spectrum-level FDR no bigger than 1%, 351 and 310 N-glycans were identified for HepG2 and LO2, respectively, with comprehensive structural information (not only monosaccharide composition, but also sequence and linkage) by N-glycan database search engine GlySeeker. The percentage of hybrid N-glycans with tetra-antennary structures was substantially increased in the HepG2 cells. This comprehensive discovery study of differentially expressed cell-surface N-glycans in HepG2 vs LO2 serves as a solid reference for future validation study of glycosylation markers in HCC.
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PMID:Comparative Glycomics Study of Cell-Surface N-Glycomes of HepG2 versus LO2 Cell Lines. 3034 78