pGlyco: a pipeline for the identification of intact N-glycopeptides by using HCD- and CID-MS/MS and MS3

Zeng, Wen‐Feng; Liu, Mingqi; Zhang, Yang; Wu, Jianqiang; Pan, Fang; Peng, Chao; Nie, Aiying; Yan, Guoquan; Cao, Weiqian; Liu, Chao; Chi, Hao; Sun, Rui-Xiang; Wong, Catherine C. L.; He, Shan; Yang, Pengyuan

doi:10.1038/srep25102

Cited by 90 publications

(105 citation statements)

References 32 publications

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“…In the MS/MS-HCD spectra, Y ions are normally quite abundant. 28 We could use these ions in addition to the b and y ions to check whether they were co-eluted and compare the rank to that recorded in the above-mentioned library ( Figure 4C). If so, we could undoubtedly pinpoint the chromatographic peak of the precursor and use the maximum intensity of the top three isotopes to represent the abundance of the glycopeptides ( Figure 4D).…”

Section: Targeted Monitoring Of Intact Glycopeptides Across Lactationmentioning

confidence: 99%

Quantitative Longitudinal Inventory of the N-Glycoproteome of Human Milk from a Single Donor Reveals the Highly Variable Repertoire and Dynamic Site-Specific Changes

et al. 2020

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Protein N-glycosylation on human milk proteins assists in protecting an infant's health and functions among others as competitive inhibitors of pathogen binding and immunomodulators. Due to the individual uniqueness of each mother's milk and the overall complexity and temporal changes of protein N-glycosylation, analysis of the human milk N-glycoproteome requires longitudinal personalized approaches, providing protein-and N-site-specific quantitative information. Here, we describe an automated platform using hydrophilic-interaction chromatography (HILIC)-based cartridges enabling the proteome-wide monitoring of intact Nglycopeptides using just a digest of 150 μg of breast milk protein. We were able to map around 1700 glycopeptides from 110 glycoproteins covering 191 glycosites, of which 43 sites have not been previously reported with experimental evidence. We next quantified 287 of these glycopeptides originating from 50 glycoproteins using a targeted proteomics approach. Although each glycoprotein, N-glycosylation site, and attached glycan revealed distinct dynamic changes, we did observe a few general trends. For instance, fucosylation, especially terminal fucosylation, increased across the lactation period. Building on the improved glycoproteomics approach outlined above, future studies are warranted to reveal the potential impact of the observed glycosylation microheterogeneity on the healthy development of infants.

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Section: Targeted Monitoring Of Intact Glycopeptides Across Lactationmentioning

confidence: 99%

Quantitative Longitudinal Inventory of the N-Glycoproteome of Human Milk from a Single Donor Reveals the Highly Variable Repertoire and Dynamic Site-Specific Changes

et al. 2020

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“…Data-dependent acquisition of MS3 for the most intense peaks of HCD-MS/MS was used to provide fragments to identify the peptide backbones. 46) In this "Glycopeptide MS/MS Data" section, "GlycoPeptide Search (GPS)" can be downloaded from https://edwardslab.bmcb.georgetown.edu/trac/GlycoPeptideSearch/. It is also a free so ware for glycopeptide tandem mass spectrum database search.…”

Section: Glycopeptide Ms/ms Datamentioning

confidence: 99%

A Brief Review of Bioinformatics Tools for Glycosylation Analysis by Mass Spectrometry

Tsai

Chen

2017

Mass Spectrometry

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e purpose of this review is to provide updated information regarding bioinformatic so ware for the use in the characterization of glycosylated structures since 2013. A comprehensive review by Woodin et al. Analyst 138: 2793Analyst 138: -2803Analyst 138: , 2013 described two main approaches that are introduced for starting researchers in this area; analysis of released glycans and the identi cation of glycopeptide in enzymatic digests, respectively. Complementary to that report, this review focuses on mass spectrometry related bioinformatics tools for the characterization of N-linked and O-linked glycopeptides. Speci cally, it also provides information regarding automated tools that can be used for glycan pro ling using mass spectrometry.

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“…It doesn't support highthroughput searching. Another software tool, pGlyco, can be utilized for intact N-linked glycopeptide analysis with an FDR estimation 31,32 . The most recent version eliminated the dependency on the multiple fragmentation strategies (i.e.…”

Section: Discussionmentioning

confidence: 99%

“…However, information related to both glycosites and the attached glycans at specific glycosylation sites need be determined. More recently, analyses of intact glycopeptides have been used to study glycosylation heterogeneity from a specific glycoprotein or complex samples [18][19][20][21][22][23][24] Recently, several software tools 25 have been developed to assist the assignment of glycopeptides, such as Byonic 26 , Glycopeptide Search (GPS) 27 , Glycopep Grader 28 , SpectraST 29 , GPQuest 30 , and pGlyco 31,32 , allowing for high throughput identification of intact glycopeptides.…”

Section: Introductionmentioning

confidence: 99%

Reanalysis of global proteomic and phosphoproteomic data identified a large number of glycopeptides

Shah

Clark

et al. 2017

Preprint

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Protein glycosylation plays fundamental roles in many cellular processes, and previous reports have shown dysregulation to be associated with several human diseases, including diabetes, cancer, and neurodegenerative disorders. Despite the vital role of glycosylation for proper protein function, the analysis of glycoproteins has been lagged behind to other protein modifications. In this study, we describe the re-analysis of global proteomic data from breast cancer xenograft tissues using recently developed software package GPQuest 2.0, revealing a large number of previously unidentified N-linked glycopeptides. More importantly, we found that using immobilized metal affinity chromatography (IMAC) technology for the enrichment of phosphopeptides had coenriched a substantial number of sialoglycopeptides, allowing for a large-scale analysis of sialoglycopeptides in conjunction with the analysis of phosphopeptides. Collectively, combined MS/MS analyses of global proteomic and phosphoproteomic datasets resulted in the identification of 6,724 N-linked glycopeptides from 617 glycoproteins derived from two breast cancer xenograft tissues. Next, we utilized GPQuest for the re-analysis of global and phosphoproteomic data generated from 108 human breast cancer tissues that were previously analyzed by Clinical Proteomic Analysis Consortium (CPTAC). Reanalysis of the CPTAC dataset resulted in the identification of 2,683 glycopeptides from the global proteomic data set and 4,554 glycopeptides from phosphoproteomic data set, respectively. Together, 11,292 N-linked glycopeptides corresponding to 1,731 N-linked glycosites from 883 human glycoproteins were identified from the two data sets. This analysis revealed an extensive number of glycopeptides hidden in the global and enriched in IMAC-based phosphopeptide-enriched proteomic data, information which would have remained unknown from the original study otherwise. The reanalysis described herein can be readily applied to identify glycopeptides from already existing data sets, providing insight into many important facets of protein glycosylation in different biological, physiological, and pathological processes.would not contain the sialic residue. Further investigation of the influence of IMAC-enrichment on intact glycopeptides, specifically sialylated intact glycopeptides is warranted, as well as the scheme of the co-enrichment of both phosphopeptides and intact glycopeptides. ConclusionsWith the rapid development of mass spectrometry and computational glycoproteomics tools, we can conduct a large-scale analysis of glycoproteome without specific glycopeptide enrichment and mass spectrometry analysis. In this study, we used our recently developed and improved intact glycopeptide analysis tool, GPQuest 2.0, to investigate the glycopeptide expression in two large datasets: two breast cancer xenograft samples and 108 CPTAC breast cancer tissues. The search results of 'oxo-spectra' and intact N-linked glycopeptides analysis demonstrated the feasibility of profiling glycoproteome utiliz...

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pGlyco: a pipeline for the identification of intact N-glycopeptides by using HCD- and CID-MS/MS and MS3

Cited by 90 publications

References 32 publications

Quantitative Longitudinal Inventory of the N-Glycoproteome of Human Milk from a Single Donor Reveals the Highly Variable Repertoire and Dynamic Site-Specific Changes

Quantitative Longitudinal Inventory of the N-Glycoproteome of Human Milk from a Single Donor Reveals the Highly Variable Repertoire and Dynamic Site-Specific Changes

A Brief Review of Bioinformatics Tools for Glycosylation Analysis by Mass Spectrometry

Reanalysis of global proteomic and phosphoproteomic data identified a large number of glycopeptides

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