Site-Specific Glycan-Peptide Analysis for Determination of N‑Glycoproteome Heterogeneity
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–tan...
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Veröffentlicht in: | Journal of proteome research 2013-12, Vol.12 (12), p.5791-5800 |
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creator | Parker, Benjamin L Thaysen-Andersen, Morten Solis, Nestor Scott, Nichollas E Larsen, Martin R Graham, Mark E Packer, Nicolle H Cordwell, Stuart J |
description | 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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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.</description><identifier>ISSN: 1535-3893</identifier><identifier>EISSN: 1535-3907</identifier><identifier>DOI: 10.1021/pr400783j</identifier><identifier>PMID: 24090084</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Amino Acid Sequence ; Animals ; Brain - metabolism ; Brain Chemistry ; Carbohydrate Sequence ; Chromatography, Liquid - instrumentation ; Chromatography, Liquid - methods ; Databases, Factual ; Genetic Heterogeneity ; Glycomics - instrumentation ; Glycomics - methods ; Glycosylation ; Humans ; Hydrogen-Ion Concentration ; Male ; Molecular Sequence Annotation ; Molecular Sequence Data ; Peptide Mapping - methods ; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - chemistry ; Protein Processing, Post-Translational ; Proteome - analysis ; Proteome - chemistry ; Rats ; Rats, Inbred Lew ; Tandem Mass Spectrometry</subject><ispartof>Journal of proteome research, 2013-12, Vol.12 (12), p.5791-5800</ispartof><rights>Copyright © 2013 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a315t-cc2492dda3cc683149305a9c9d27464dcf09f0a316dfd0a21d405b4b892811a23</citedby><cites>FETCH-LOGICAL-a315t-cc2492dda3cc683149305a9c9d27464dcf09f0a316dfd0a21d405b4b892811a23</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/pr400783j$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/pr400783j$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24090084$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Parker, Benjamin L</creatorcontrib><creatorcontrib>Thaysen-Andersen, Morten</creatorcontrib><creatorcontrib>Solis, Nestor</creatorcontrib><creatorcontrib>Scott, Nichollas E</creatorcontrib><creatorcontrib>Larsen, Martin R</creatorcontrib><creatorcontrib>Graham, Mark E</creatorcontrib><creatorcontrib>Packer, Nicolle H</creatorcontrib><creatorcontrib>Cordwell, Stuart J</creatorcontrib><title>Site-Specific Glycan-Peptide Analysis for Determination of N‑Glycoproteome Heterogeneity</title><title>Journal of proteome research</title><addtitle>J. Proteome Res</addtitle><description>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.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Brain - metabolism</subject><subject>Brain Chemistry</subject><subject>Carbohydrate Sequence</subject><subject>Chromatography, Liquid - instrumentation</subject><subject>Chromatography, Liquid - methods</subject><subject>Databases, Factual</subject><subject>Genetic Heterogeneity</subject><subject>Glycomics - instrumentation</subject><subject>Glycomics - methods</subject><subject>Glycosylation</subject><subject>Humans</subject><subject>Hydrogen-Ion Concentration</subject><subject>Male</subject><subject>Molecular Sequence Annotation</subject><subject>Molecular Sequence Data</subject><subject>Peptide Mapping - methods</subject><subject>Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - chemistry</subject><subject>Protein Processing, Post-Translational</subject><subject>Proteome - analysis</subject><subject>Proteome - chemistry</subject><subject>Rats</subject><subject>Rats, Inbred Lew</subject><subject>Tandem Mass Spectrometry</subject><issn>1535-3893</issn><issn>1535-3907</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpt0LtOwzAUBmALgWgpDLwAyoIEQ-D4kovHqkCLVAFSYWGJXNtBrpI42M7QjVfgFXkSUrV0Yjpn-M6vox-hcww3GAi-bR0DyHK6OkBDnNAkphyyw78953SATrxfAeAkA3qMBoQBB8jZEL0vTNDxotXSlEZG02otRRO_6DYYpaNxI6q1Nz4qrYvudNCuNo0IxjaRLaOnn6_vzYFtnQ3a1jqabYj90I02YX2KjkpReX22myP09nD_OpnF8-fp42Q8jwXFSYilJIwTpQSVMs0pZpxCIrjkimQsZUqWwEvobapKBYJgxSBZsmXOSY6xIHSErra5_RufnfahqI2XuqpEo23nC8zSlGYJyWhPr7dUOuu902XROlMLty4wFJsqi32Vvb3YxXbLWqu9_OuuB5dbIKQvVrZzfVn-n6Bfhxx7sg</recordid><startdate>20131206</startdate><enddate>20131206</enddate><creator>Parker, Benjamin L</creator><creator>Thaysen-Andersen, Morten</creator><creator>Solis, Nestor</creator><creator>Scott, Nichollas E</creator><creator>Larsen, Martin R</creator><creator>Graham, Mark E</creator><creator>Packer, Nicolle H</creator><creator>Cordwell, Stuart J</creator><general>American Chemical Society</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20131206</creationdate><title>Site-Specific Glycan-Peptide Analysis for Determination of N‑Glycoproteome Heterogeneity</title><author>Parker, Benjamin L ; Thaysen-Andersen, Morten ; Solis, Nestor ; Scott, Nichollas E ; Larsen, Martin R ; Graham, Mark E ; Packer, Nicolle H ; Cordwell, Stuart J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a315t-cc2492dda3cc683149305a9c9d27464dcf09f0a316dfd0a21d405b4b892811a23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Brain - metabolism</topic><topic>Brain Chemistry</topic><topic>Carbohydrate Sequence</topic><topic>Chromatography, Liquid - instrumentation</topic><topic>Chromatography, Liquid - methods</topic><topic>Databases, Factual</topic><topic>Genetic Heterogeneity</topic><topic>Glycomics - instrumentation</topic><topic>Glycomics - methods</topic><topic>Glycosylation</topic><topic>Humans</topic><topic>Hydrogen-Ion Concentration</topic><topic>Male</topic><topic>Molecular Sequence Annotation</topic><topic>Molecular Sequence Data</topic><topic>Peptide Mapping - methods</topic><topic>Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - chemistry</topic><topic>Protein Processing, Post-Translational</topic><topic>Proteome - analysis</topic><topic>Proteome - chemistry</topic><topic>Rats</topic><topic>Rats, Inbred Lew</topic><topic>Tandem Mass Spectrometry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Parker, Benjamin L</creatorcontrib><creatorcontrib>Thaysen-Andersen, Morten</creatorcontrib><creatorcontrib>Solis, Nestor</creatorcontrib><creatorcontrib>Scott, Nichollas E</creatorcontrib><creatorcontrib>Larsen, Martin R</creatorcontrib><creatorcontrib>Graham, Mark E</creatorcontrib><creatorcontrib>Packer, Nicolle H</creatorcontrib><creatorcontrib>Cordwell, Stuart J</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of proteome research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Parker, Benjamin L</au><au>Thaysen-Andersen, Morten</au><au>Solis, Nestor</au><au>Scott, Nichollas E</au><au>Larsen, Martin R</au><au>Graham, Mark E</au><au>Packer, Nicolle H</au><au>Cordwell, Stuart J</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Site-Specific Glycan-Peptide Analysis for Determination of N‑Glycoproteome Heterogeneity</atitle><jtitle>Journal of proteome research</jtitle><addtitle>J. Proteome Res</addtitle><date>2013-12-06</date><risdate>2013</risdate><volume>12</volume><issue>12</issue><spage>5791</spage><epage>5800</epage><pages>5791-5800</pages><issn>1535-3893</issn><eissn>1535-3907</eissn><abstract>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.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>24090084</pmid><doi>10.1021/pr400783j</doi><tpages>10</tpages></addata></record> |
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subjects | Amino Acid Sequence Animals Brain - metabolism Brain Chemistry Carbohydrate Sequence Chromatography, Liquid - instrumentation Chromatography, Liquid - methods Databases, Factual Genetic Heterogeneity Glycomics - instrumentation Glycomics - methods Glycosylation Humans Hydrogen-Ion Concentration Male Molecular Sequence Annotation Molecular Sequence Data Peptide Mapping - methods Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - chemistry Protein Processing, Post-Translational Proteome - analysis Proteome - chemistry Rats Rats, Inbred Lew Tandem Mass Spectrometry |
title | Site-Specific Glycan-Peptide Analysis for Determination of N‑Glycoproteome Heterogeneity |
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