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<title>Identification of intact glycopeptides by liquid chromatography/tandem mass spectrometry followed by database search - Glycoscience Protocols (GlycoPODv2) - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Glycoscience Protocols (GlycoPODv2) [Internet]" /><meta name="citation_title" content="Identification of intact glycopeptides by liquid chromatography/tandem mass spectrometry followed by database search" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2022/03/28" /><meta name="citation_author" content="Hiroyuki Kaji" /><meta name="citation_pmid" content="37590718" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK593989/" /><meta name="citation_keywords" content="intact glycopeptide" /><meta name="citation_keywords" content="mass spectrometry (MS)" /><meta name="citation_keywords" content="high-energy collision-induced dissociation (HCD)" /><meta name="citation_keywords" content="electron-transfer dissociation (ETD)" /><meta name="citation_keywords" content="Mascot" /><meta name="citation_keywords" content="Byonic" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Identification of intact glycopeptides by liquid chromatography/tandem mass spectrometry followed by database search" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Hiroyuki Kaji" /><meta name="DC.Date" content="2022/03/28" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK593989/" /><meta name="description" content="The mass spectrometric analysis of intact glycopeptides is structurally difficult because glycopeptides are composed of different oligomeric compounds, peptides, and oligosaccharide(s). To identify peptides in common MS method, partial fragmentation of (preferably single) peptide bond by e.g., collision-induced dissociation (CID) followed by the acquisition of the fragment mass spectrum (MS/MS or MS2 spectrum) is used. However, glycoside bonds between peptide and glycan and between monosaccharides are weaker than the peptide bond; thus, glycoside bonds are preferentially cleaved by CID and peptide bonds are not cleaved, thereby peptide portion could not be identified. With the first CID of N-glycopeptide ion selected, fragment ions of peptide (called Y0) and peptide having a single inner GlcNAc of chitobiose core (Y1) are often generated; thus, by selecting one of these ions to cleave by 2nd CID, peptide fragment ions will be generated. By analyzing the fragment spectrum (MS/MS/MS or MS3), peptide portion might be assignable (Figure 1). Currently, high-energy CID (HCD) can cleave peptide and glycoside bonds simultaneously. Using the HCD MS2 spectrum, intact glycopeptides can be identified at improved sensitivity (Figure 1). Several software for the identification of glycopeptides based on the HCD spectrum are available and are developing actively (1). For O-glycopeptides having relatively short glycans, such as Tn-antigen (O-GalNAc), T-antigen (core 1; Gal-GalNAc), sialyl T, disialyl T, etc, their identification is possible using HCD MS2, and glycosylated site is also assignable using electron-transfer dissociation (ETD) MS2, as described elsewhere in this protocol series, GlycoPOD. With the Orbitrap Fusion tribrid mass spectrometer, more powerful acquisition of MS2, such as HCD fragment-triggered HCD/ETD/EThcD/ETciD spectra, are available. In this section, the method for identifying N-glycopeptide with HCD MS2 spectra is introduced." /><meta name="og:title" content="Identification of intact glycopeptides by liquid chromatography/tandem mass spectrometry followed by database search" /><meta name="og:type" content="book" /><meta name="og:description" content="The mass spectrometric analysis of intact glycopeptides is structurally difficult because glycopeptides are composed of different oligomeric compounds, peptides, and oligosaccharide(s). To identify peptides in common MS method, partial fragmentation of (preferably single) peptide bond by e.g., collision-induced dissociation (CID) followed by the acquisition of the fragment mass spectrum (MS/MS or MS2 spectrum) is used. However, glycoside bonds between peptide and glycan and between monosaccharides are weaker than the peptide bond; thus, glycoside bonds are preferentially cleaved by CID and peptide bonds are not cleaved, thereby peptide portion could not be identified. With the first CID of N-glycopeptide ion selected, fragment ions of peptide (called Y0) and peptide having a single inner GlcNAc of chitobiose core (Y1) are often generated; thus, by selecting one of these ions to cleave by 2nd CID, peptide fragment ions will be generated. By analyzing the fragment spectrum (MS/MS/MS or MS3), peptide portion might be assignable (Figure 1). Currently, high-energy CID (HCD) can cleave peptide and glycoside bonds simultaneously. Using the HCD MS2 spectrum, intact glycopeptides can be identified at improved sensitivity (Figure 1). Several software for the identification of glycopeptides based on the HCD spectrum are available and are developing actively (1). For O-glycopeptides having relatively short glycans, such as Tn-antigen (O-GalNAc), T-antigen (core 1; Gal-GalNAc), sialyl T, disialyl T, etc, their identification is possible using HCD MS2, and glycosylated site is also assignable using electron-transfer dissociation (ETD) MS2, as described elsewhere in this protocol series, GlycoPOD. With the Orbitrap Fusion tribrid mass spectrometer, more powerful acquisition of MS2, such as HCD fragment-triggered HCD/ETD/EThcD/ETciD spectra, are available. In this section, the method for identifying N-glycopeptide with HCD MS2 spectra is introduced." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK593989/" /><meta name="og:site_name" content="NCBI Bookshelf" /><meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-glycopodv2-lrg.png" /><meta name="twitter:card" content="summary" /><meta name="twitter:site" content="@ncbibooks" /><meta name="bk-non-canon-loc" content="/books/n/glycopodv2/g139-massdatabase/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK593989/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} </style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script>
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK593989_"><span class="title" itemprop="name">Identification of intact glycopeptides by liquid chromatography/tandem mass spectrometry followed by database search</span></h1><div class="contrib half_rhythm"><span itemprop="author">Hiroyuki Kaji</span>, Dr.<div class="affiliation small">iGCORE, Nagoya University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-ayogan.dem@ikuyorih-ijak" class="oemail">pj.ca.u-ayogan.dem@ikuyorih-ijak</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">October 1, 2021</span>; Last Revision: <span itemprop="dateModified">March 28, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g139-massdatabase.Introduction"><h2 id="_g139-massdatabase_Introduction_">Introduction</h2><p>The mass spectrometric analysis of intact glycopeptides is structurally difficult because glycopeptides are composed of different oligomeric compounds, peptides, and oligosaccharide(s). To identify peptides in common MS method, partial fragmentation of (preferably single) peptide bond by e.g., collision-induced dissociation (CID) followed by the acquisition of the fragment mass spectrum (MS/MS or MS<sup>2</sup> spectrum) is used. However, glycoside bonds between peptide and glycan and between monosaccharides are weaker than the peptide bond; thus, glycoside bonds are preferentially cleaved by CID and peptide bonds are not cleaved, thereby peptide portion could not be identified. With the first CID of <i>N</i>-glycopeptide ion selected, fragment ions of peptide (called Y0) and peptide having a single inner GlcNAc of chitobiose core (Y1) are often generated; thus, by selecting one of these ions to cleave by 2<sup>nd</sup> CID, peptide fragment ions will be generated. By analyzing the fragment spectrum (MS/MS/MS or MS<sup>3</sup>), peptide portion might be assignable (<a class="figpopup" href="/books/NBK593989/figure/g139-massdatabase.F1/?report=objectonly" target="object" rid-figpopup="figg139massdatabaseF1" rid-ob="figobg139massdatabaseF1">Figure 1</a>). Currently, high-energy CID (HCD) can cleave peptide and glycoside bonds simultaneously. Using the HCD MS<sup>2</sup> spectrum, intact glycopeptides can be identified at improved sensitivity (<a class="figpopup" href="/books/NBK593989/figure/g139-massdatabase.F1/?report=objectonly" target="object" rid-figpopup="figg139massdatabaseF1" rid-ob="figobg139massdatabaseF1">Figure 1</a>). Several software for the identification of glycopeptides based on the HCD spectrum are available and are developing actively (<a class="bk_pop" href="#g139-massdatabase.REF.1">1</a>). For <i>O</i>-glycopeptides having relatively short glycans, such as Tn-antigen (<i>O</i>-GalNAc), T-antigen (core 1; Gal-GalNAc), sialyl T, disialyl T, etc, their identification is possible using HCD MS<sup>2</sup>, and glycosylated site is also assignable using electron-transfer dissociation (ETD) MS<sup>2</sup>, as described elsewhere in this protocol series, GlycoPOD. With the Orbitrap Fusion tribrid mass spectrometer, more powerful acquisition of MS<sup>2</sup>, such as HCD fragment-triggered HCD/ETD/EThcD/ETciD spectra, are available. In this section, the method for identifying <i>N</i>-glycopeptide with HCD MS<sup>2</sup> spectra is introduced.</p></div><div id="g139-massdatabase.Protocol"><h2 id="_g139-massdatabase_Protocol_">Protocol</h2><p>This chapter describes a protocol for identifying intact glycopeptides in a complex glycopeptide mixture using two database search engines, Mascot and Byonic, based on MS/MS spectra acquired using liquid chromatography–mass spectrometry (LC-MS) analysis. As described in Introduction, a series of MS/MS-based search engine software have been developed (<a class="bk_pop" href="#g139-massdatabase.REF.1">1</a>). Byonic showed a good performance in the comparative study.</p><div id="g139-massdatabase.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Glycopeptide sample</p></dd></dl></div><div id="g139-massdatabase.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">LC-MS system: Nanoflow LC: Ultimate 3000 (Thermo Fisher Scientific, Waltham, MA, US)</p></dd><dt>2.</dt><dd><p class="no_top_margin">LC-MS system: Electrospray ionization (ESI)–tandem mass spectrometer equipped HCD: Orbitrap Fusion tribrid mass spectrometer (Thermo Fisher Scientific)</p></dd><dt>3.</dt><dd><p class="no_top_margin">Database search engine: Mascot server (Ver.2.6.2, Matrix Science, Boston, MA, US)/Byonic (Protein Metrics, Cupertino, CA, US)</p></dd></dl></div><div id="g139-massdatabase.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Preparation of a peptide or glycopeptide sample</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Prepare a peptide or a glycopeptide sample as described in other sections in this series.</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">LC-MS analysis of (glyco)peptide sample</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Analyze the (glyco)peptide sample using LC–ESI–tandem mass spectrometer equipped HCD.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Representative LC-MS conditions: Mode: positive; MS<sup>1</sup> detector: Orbitrap; MS<sup>1</sup> resolution: 120,000 at m/z 200; MS<sup>1</sup> mass range: 400–2,000; Activation: HCD; Normalized collision energy: 30%; Data acquisition: data-dependent or data-dependent HCD fragment-triggered HCD acquisition; Trigger: 204.0872 (HexNAc+H<sup>+</sup>); MS<sup>2</sup> detector: Orbitrap; MS<sup>2</sup> resolution: 15,000; and MS<sup>2</sup> first mass: 135.</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Database search by Mascot server</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Convert the MS raw data to Mascot generic format (MGF) using Mascot Distiller, which is performed by Mascot Daemon, and then MS<sup>2</sup> spectra are deconvoluted.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Search HCD MS<sup>2</sup> spectra (MGF) using the Mascot server and any appropriate protein sequence database. The number of assuming variable modifications (glycan composition) should be one or two in a single search, e.g., M5 (HexNAc2Hex5), hybrid (HexNAc3Hex6), or biantennary (HexNAc4Hex5). Then, the setting of glycosylation should be modified considering neutral losses and ignore masses since CID may cause partial neutral losses of mono- or oligosaccharides and generate glycan fragments, which have no means for peptide assignment. For example, in the case of HexNAc4Hex5, set neutral losses of Hex, Hex2, Hex2HexNAc1, Hex2HexNAc2, …, Hex5HexNAc3, and Hex5HexNAc4 and ignore masses, such as H+HexNAc (204.0872), H+Hex1HexNAc1 (366.1400), etc. (<b>Note 1</b>)</p></dd><dt>c.</dt><dd><p class="no_top_margin">Representative search conditions: Method: MS/MS ion search; Database: SwissProt_UniProtKB_isoform; Enzyme: Trypsin; Fixed modifications: Carbamidomethy (Cys); Variable modifications: Ammonia-loss (N-term Cys), Gln->pyroGlu (N-term Gln), Oxidation (Met), and glycosylation, e.g., Hex5HexNAc2 (modified in neutral losses and ignore masses); Peptide mass tolerance: 7 ppm; Fragment mass tolerance: 0.02 Da; Maximum missed cleavage: 2; and Instrument type: Electrospray ionization–Fourier transform ion cyclotron resonance.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Representative annotated MS<sup>2</sup> spectra are shown in <a class="figpopup" href="/books/NBK593989/figure/g139-massdatabase.F2/?report=objectonly" target="object" rid-figpopup="figg139massdatabaseF2" rid-ob="figobg139massdatabaseF2">Figures 2</a> and <a class="figpopup" href="/books/NBK593989/figure/g139-massdatabase.F3/?report=objectonly" target="object" rid-figpopup="figg139massdatabaseF3" rid-ob="figobg139massdatabaseF3">3</a>.</p></dd></dl></dd><dt>4.</dt><dd><p class="no_top_margin">Database search using Byonic</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Byonic search is performed via Proteome Discoverer using MS raw data.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Set the workflow of the Proteome Discoverer to use Spectrum Selector.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Representative search conditions: Database: SwissProt_UniProtKB_isoform; Enzyme: Trypsin_KR (full); Maximum missed cleavage: 2; Precursor mass tolerance: 7 ppm; Fragmentation type: HCD; Fragment mass tolerance: 0.02 Da; Modifications: Static: Carbamidomethy (Cys) and Dynamic: Ammonia-loss (<i>N</i>-term Cys), Gln->pyroGlu (<i>N</i>-term Gln), and Oxidation (Met). For Byonic, the dynamic modification can be set as either “common” or “rare,” and these have separate limit numbers of occurrences per single peptide. Please set an appropriate category and a limit number for each modification according to the rules of the software. Searching with numerous modifications for O-glycans takes time; and Glycan database: e.g., <i>N</i>-glycan 132 human.</p></dd><dt>d.</dt><dd><p class="no_top_margin">A representative annotated MS<sup>2</sup> spectrum is shown in <a class="figpopup" href="/books/NBK593989/figure/g139-massdatabase.F4/?report=objectonly" target="object" rid-figpopup="figg139massdatabaseF4" rid-ob="figobg139massdatabaseF4">Figure 4</a>. (<b>Note 2</b>).</p></dd></dl></dd></dl></div><div id="g139-massdatabase.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">On Mascot search, it is not preferable to set many glycan compositions as variable modifications. Thus, searching and selecting one or two compositions is recommended. Furthermore, customizing modifications, including neutral losses and ignore masses, is important for increasing the possibility of identification. If reliable identification is obtained, glycopeptide ions having a common peptide, but different glycan compositions may exist at near-retention time of the identified glycopeptide.</p></dd><dt>2.</dt><dd><p class="no_top_margin">Byonic is a powerful search engine as it can search while considering numerous glycan compositions. However, it has been indicated that it is difficult to set criteria of certainty (<a class="bk_pop" href="#g139-massdatabase.REF.2">2</a>). It may be necessary to confirm the presence of glycan diagnostic ions, match of the Y0/Y1 ions with the identified peptide, and partial sequence inspection.</p></dd></dl></div></div><div id="g139-massdatabase.References"><h2 id="_g139-massdatabase_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g139-massdatabase.REF.1">Kawahara R, Chernykh A, Alagesan K, Bern M, Cao W, Chalkley RJ, Cheng K, Choo MS, Edwards N, Goldman R, Hoffmann M, Hu Y, Huang Y, Kim JY, Kletter D, Liquet B, Liu M, Mechref Y, Meng B, Neelamegham S, Nguyen-Khuong T, Nilsson J, Pap A, Park GW, Parker BL, Pegg CL, Penninger JM, Phung TK, Pioch M, Rapp E, Sakalli E, Sanda M, Schulz BL, Scott NE, Sofronov G, Stadlmann J, Vakhrushev SY, Woo CM, Wu HY, Yang P, Ying W, Zhang H, Zhang Y, Zhao J, Zaia J, Haslam SM, Palmisano G, Yoo JS, Larson G, Khoo KH, Medzihradszky KF, Kolarich D, Packer NH, Thaysen-Andersen M. Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis. <span><span class="ref-journal">Nat Methods. </span>2021 Nov;<span class="ref-vol">18</span>(11):1304–1316.</span> [<a href="/pmc/articles/PMC8566223/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC8566223</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/34725484" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 34725484</span></a>] [<a href="http://dx.crossref.org/10.1038/s41592-021-01309-x" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g139-massdatabase.REF.2">Go EP, Zhang S, Ding H, Kappes JC, Sodroski J, Desaire H. The opportunity cost of automated glycopeptide analysis: case study profiling the SARS-CoV-2 S glycoprotein. <span><span class="ref-journal">Anal Bioanal Chem. </span>2021 Dec;<span class="ref-vol">413</span>(29):7215–7227.</span> [<a href="/pmc/articles/PMC8390178/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC8390178</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/34448030" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 34448030</span></a>] [<a href="http://dx.crossref.org/10.1007/s00216-021-03621-z" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593989_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g139-massdatabase.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div class="bk_prnt_sctn"><h2>Figures</h2><div class="whole_rhythm bk_prnt_obj bk_first_prnt_obj"><div id="g139-massdatabase.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593989/bin/g139-massdatabase-Image001.jpg" alt="Figure 1: . Acquisition of fragment mass spectrum (MS/MS spectrum) for the identification of intact N-glycopeptide using database search." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Acquisition of fragment mass spectrum (MS/MS spectrum) for the identification of intact <i>N</i>-glycopeptide using database search. The peptide portion of glycopeptide is difficult to fragment using collision-induced dissociation (CID) since glycoside bonds of glycan are weaker than peptide bonds and are broken preferentially, as shown in the upper middle spectrum. Therefore, peptide bonds are not cleaved, and peptide sequence cannot be determined. With this CID, ions of peptide and peptide remaining single GlcNAc are often generated (called Y0 and Y1 ions, respectively). If Y0/Y1 ions are selected to fragment again and fragment ion spectrum (MS/MS/MS) may be acquired, the peptide sequence becomes assignable. Currently, high-energy CID (HCD) is available, and both peptide and glycoside bonds can be fragment simultaneously. Using these MS<sup>2</sup> spectra, glycan composition and peptide sequence of intact glycopeptide can be identified using database search.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g139-massdatabase.F2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593989/bin/g139-massdatabase-Image002.jpg" alt="Figure 2: . An annotated high-energy collision-induced dissociation (HCD) MS2 spectrum of a glycopeptide carrying M5 (oligomannose-type glycan containing 5 mannoses) identified using Mascot search." /></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>An annotated high-energy collision-induced dissociation (HCD) MS<sup>2</sup> spectrum of a glycopeptide carrying M5 (oligomannose-type glycan containing 5 mannoses) identified using Mascot search. A series of y-ions were assigned, and glycan fragment ions called diagnostic ions were also detected. It is important to set ignore masses to prevent the selection of meaningless glycan-derived ions for peptide-derived ion scoring.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g139-massdatabase.F3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593989/bin/g139-massdatabase-Image003.jpg" alt="Figure 3: . An annotated high-energy collision-induced dissociation (HCD) MS2 spectrum of a glycopeptide carrying a biantennary glycan having core fucose identified using Mascot search." /></div><h3><span class="label">Figure 3: </span></h3><div class="caption"><p>An annotated high-energy collision-induced dissociation (HCD) MS<sup>2</sup> spectrum of a glycopeptide carrying a biantennary glycan having core fucose identified using Mascot search. Upper glycan structure is the most often seen structure for the composition. From this MS<sup>2</sup> spectrum, glycan composition of this glycopeptide is presumed to be Hex(5)HexNAc(4)Fuc(<a class="bk_pop" href="#g139-massdatabase.REF.1">1</a>). The presence of core fucose may be confirmed by CID MS<sup>2</sup> spectrum showing Y1+Fuc ion.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g139-massdatabase.F4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593989/bin/g139-massdatabase-Image004.jpg" alt="Figure 4: . An annotated high-energy collision-induced dissociation (HCD) MS2 spectrum of a glycopeptide carrying an M5 glycan identified using Byonic search." /></div><h3><span class="label">Figure 4: </span></h3><div class="caption"><p>An annotated high-energy collision-induced dissociation (HCD) MS<sup>2</sup> spectrum of a glycopeptide carrying an M5 glycan identified using Byonic search. Like this case, if the glycan is attached near the terminal of the peptide and many peptide-fragment ions can be assigned consecutively, the possibility of glycopeptide identification increases.</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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<div class="post-content"><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a><p class="small">Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Unported license. To view a copy of this license, visit <a href="http://creativecommons.org/licenses/by-nc-nd/4.0/" ref="pagearea=meta&targetsite=external&targetcat=link&targettype=uri">http://creativecommons.org/licenses/by-nc-nd/4.0/</a>.</p></div><div class="small"><span class="label">Bookshelf ID: NBK593989</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/37590718" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">37590718</a></span></div><div style="margin-top:2em" class="bk_noprnt"><a class="bk_cntns" href="/books/n/glycopodv2/">Contents</a><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/glycopodv2/g195-amynolysissia/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/glycopodv2/g140-Ionmobilitymass/" title="Next page in this title">Next ></a></div></div></div></div>
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