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

Ovarian cancer is a fatal gynaecological malignancy in adult women with a five-year overall survival rate of only 30%. Glycomic and glycoproteomic profiling studies have reported extensive protein glycosylation pattern alterations in ovarian cancer. Therefore, spatio-temporal investigation of these glycosylation changes may unearth tissue-specific changes that occur in the development and progression of ovarian cancer. A novel method for investigating tissue-specific N-linked glycans is using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) on formalin-fixed paraffin-embedded (FFPE) tissue sections that can spatially profile N-glycan compositions released from proteins in tissue-specific regions. In this study, tissue regions of interest (e.g. tumor, stroma, adipose tissue and necrotic areas) were isolated from FFPE tissue sections of advanced serous ovarian cancers (n = 3). PGC-LC-ESI-MS/MS and MALDI-MSI were used as complementary techniques to firstly generate structural information on the tissue-specific glycans in order to then obtain high resolution images of the glycan structure distribution in ovarian cancer tissue. The N-linked glycan repertoires carried by the proteins in these tissue regions were structurally characterized for the first time in FFPE ovarian cancer tissue regions, using enzymatic peptide-N-glycosidase F (PNGase F) release of N-glycans. The released glycans were analyzed by porous graphitized carbon liquid chromatography (PGC-LC) and collision induced electrospray negative mode MS fragmentation analysis. The N-glycan profiles identified by this analysis were then used to determine the location and distribution of each N-glycan on FFPE ovarian cancer sections that were treated with PNGase F using high resolution MALDI-MSI. A tissue-specific distribution of N-glycan structures identified particular regions of the ovarian cancer sections. For example, high mannose glycans were predominantly expressed in the tumor tissue region whereas complex/hybrid N-glycans were significantly abundant in the intervening stroma. Therefore, tumor and non-tumor tissue regions were clearly demarcated solely on their N-glycan structure distributions.
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PMID:N-glycan MALDI Imaging Mass Spectrometry on Formalin-Fixed Paraffin-Embedded Tissue Enables the Delineation of Ovarian Cancer Tissues. 2741 89

The estimation of post-mortem interval (PMI) is a crucial part for investigations of crime and untimely deaths in forensic science. However, standard methods of PMI estimation are easily confounded by extenuating circumstances and/or environmental factors. Therefore, a panel of PMI markers obtained from a more acceptable and accurate method is necessary to definitely determine time of death. Saliva, one of the vital fluids encountered at crime scenes, contains various glycoproteins that are highly affected by biochemical environment. Here, we investigated saliva N-glycans between live and dead rats to determine the alteration of N-glycans using an animal model system because of the limitation of saliva collection from recently deceased humans. Rat saliva samples were collected both before and after death. N-Glycans were enzymatically released by PNGase F without any glycoprotein extraction. Released native glycans were purified and enriched by PGC-SPE. About 100 N-glycans were identified, profiled, and structurally elucidated by nano LC/MS and tandem MS. Sialylated N-glycans were exclusively present in abundance in live rat saliva whereas non-sialylated N-glycans including LacdiNAc disaccharides were detected in high level following death. Through in-depth investigations using quantitative comparison and statistical analysis, 14 N-glycans that significantly changed after death were identified as the potential marker candidates for PMI estimation. To the best of our knowledge, this is the first study to monitor the post-mortem changes of saliva glycosylation, with obvious forensic applications.
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PMID:Monitoring of post-mortem changes of saliva N-glycosylation by nano LC/MS. 2916 81

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