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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="Preparation of glycopeptides by lectin affinity chromatography (LAC)" /><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="37590652" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK593913/" /><meta name="citation_keywords" content="glycopeptide capturing" /><meta name="citation_keywords" content="lectin affinity chromatography" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Preparation of glycopeptides by lectin affinity chromatography (LAC)" /><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/NBK593913/" /><meta name="description" content="For a wider and deeper analysis of glycopeptides using mass spectrometry, the enrichment of glycopeptides from the protease digest of the sample is effective. Especially, for identifying glycopeptide (glycoprotein) having specific glycan motif recognized by certain lectins, lectin capturing of target glycopeptides is straightforward (Note 1). The current mass spectrometric analysis of glycopeptides provides a glycan composition described with fuzzy monosaccharides, such as hexose (Hex) and N-acetylhexosamine (HexNAc), and peptide sequence. From the mass value alone, it is not possible to distinguish whether Hex is glucose, galactose, or mannose and HexNAc is GlcNAc or GalNAc. However, if sample glycopeptides are reactive with a certain lectin, a motif on the glycan might be presumable from the specificity of the lectin. In case of the lectin used has a relatively strong affinity for glycan through monovalent interaction, the lectin will be applicable to affinity chromatography (Note 2)." /><meta name="og:title" content="Preparation of glycopeptides by lectin affinity chromatography (LAC)" /><meta name="og:type" content="book" /><meta name="og:description" content="For a wider and deeper analysis of glycopeptides using mass spectrometry, the enrichment of glycopeptides from the protease digest of the sample is effective. Especially, for identifying glycopeptide (glycoprotein) having specific glycan motif recognized by certain lectins, lectin capturing of target glycopeptides is straightforward (Note 1). The current mass spectrometric analysis of glycopeptides provides a glycan composition described with fuzzy monosaccharides, such as hexose (Hex) and N-acetylhexosamine (HexNAc), and peptide sequence. From the mass value alone, it is not possible to distinguish whether Hex is glucose, galactose, or mannose and HexNAc is GlcNAc or GalNAc. However, if sample glycopeptides are reactive with a certain lectin, a motif on the glycan might be presumable from the specificity of the lectin. In case of the lectin used has a relatively strong affinity for glycan through monovalent interaction, the lectin will be applicable to affinity chromatography (Note 2)." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK593913/" /><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/g6-prepchroma/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK593913/" /><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="_NBK593913_"><span class="title" itemprop="name">Preparation of glycopeptides by lectin affinity chromatography (LAC)</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><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="g6-prepchroma.Introduction"><h2 id="_g6-prepchroma_Introduction_">Introduction</h2><p>For a wider and deeper analysis of glycopeptides using mass spectrometry, the enrichment of glycopeptides from the protease digest of the sample is effective. Especially, for identifying glycopeptide (glycoprotein) having specific glycan motif recognized by certain lectins, lectin capturing of target glycopeptides is straightforward (<b>Note 1</b>). The current mass spectrometric analysis of glycopeptides provides a glycan composition described with fuzzy monosaccharides, such as hexose (Hex) and <i>N</i>-acetylhexosamine (HexNAc), and peptide sequence. From the mass value alone, it is not possible to distinguish whether Hex is glucose, galactose, or mannose and HexNAc is GlcNAc or GalNAc. However, if sample glycopeptides are reactive with a certain lectin, a motif on the glycan might be presumable from the specificity of the lectin. In case of the lectin used has a relatively strong affinity for glycan through monovalent interaction, the lectin will be applicable to affinity chromatography (<b>Note 2</b>).</p></div><div id="g6-prepchroma.Protocol"><h2 id="_g6-prepchroma_Protocol_">Protocol</h2><p>This chapter describes a protocol that uses a lectin, <i>Aleuria aurantia</i> lectin (AAL), as an example (<b>Note 3</b>). Since this lectin has a relatively high binding affinity for glycans and glycopeptides, it can be applied to capture glycopeptides through their monovalent interaction. When selecting a lectin as an affinity ligand, the lectin should have sufficient binding capacity to retain glycopeptides during loading and washing and the elution conditions should be known.</p><div id="g6-prepchroma.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Empty column; e.g., Tricorn 5/50 column (Cytiva, Marlborough, MA, US)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Lectin-immobilized gel; e.g., AAL, Agarose Bound (Vector Laboratories, Burlingame, CA, US)</p></dd><dt>3.</dt><dd><p class="no_top_margin">Elution sugar; e.g., for AAL, α-L-Fucose (Sigma-Aldrich, Saint Louis, MO, US)</p></dd><dt>4.</dt><dd><p class="no_top_margin">Equilibration buffer for AAL; 10 mM of HEPES-NaOH, pH 7.5</p></dd><dt>5.</dt><dd><p class="no_top_margin">Elution buffer for AAL; 20 mM of α-L-Fucose/equilibration buffer</p></dd><dt>6.</dt><dd><p class="no_top_margin">C18 column; Mightysil RP-18GP (2 mm id × 50 mm, Kanto Chemical, Tokyo, Japan)</p></dd><dt>7.</dt><dd><p class="no_top_margin">Acetonitrile (MeCN, Merck, Kenilworth, NJ, US)</p></dd><dt>8.</dt><dd><p class="no_top_margin">Trifluoroacetic acid (TFA, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)</p></dd></dl></div><div id="g6-prepchroma.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">High-performance liquid chromatography system</p></dd></dl></div><div id="g6-prepchroma.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Preparation of a protease digest of a protein sample</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Prepare a protease digest of a sample protein, e.g., as described in the other section.</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Capturing of glycopeptide using lectin affinity chromatography</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Prepare a lectin column packed with lectin-immobilized gel according to the procedure recommended by the manufacturer.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Wash the column with an elution buffer (containing elution sugar [e.g., for AAL, 20 mM of L-fucose] and 5 volume of the gel) and equilibrate the column monitoring absorbance at 230 nm.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Apply sample peptides (protease digest treated with protease inhibitor to protect lectin ligand) on the column and recover the pass-through.</p></dd><dt>d.</dt><dd><p class="no_top_margin">After washing, elute glycopeptides by injecting the elution buffer and collect the eluted glycopeptides. A representative elution profile is shown in <a class="figpopup" href="/books/NBK593913/figure/g6-prepchroma.F1/?report=objectonly" target="object" rid-figpopup="figg6prepchromaF1" rid-ob="figobg6prepchromaF1">Figure 1</a>.</p></dd><dt>e.</dt><dd><p class="no_top_margin">To remove sugar and buffering chemicals, after acidification of the glycopeptide solution with diluted TFA to pH 2, apply it to the C18 reverse-phase column equilibrated with 0.1% TFA.</p></dd><dt>f.</dt><dd><p class="no_top_margin">After washing, elute the glycopeptides with 50% MeCN containing 0.1% TFA.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Evaporate the solvent from the eluate.</p></dd></dl></dd></dl></div><div id="g6-prepchroma.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Presently, it is widely recognized that glycosylation status, such as glycan structure, occupancy, and site alter associated with change of cell state, e.g., differentiation and carcinogenesis. Thus, protein carrying glycans emerged by the disease onset is specific to the disease cells, suggesting that the glycoprotein is a potential biomarker. Lectin microarray analysis is a sensitive and powerful method for detecting such disease-related glycan changes, and the results directly provide a series of representative lectins reflecting the change well. Therefore, lectin affinity capture using the reflecting lectin followed by the identification of the bound peptides using mass spectrometry is a reasonable pipeline to discover disease biomarker candidates (<a class="bk_pop" href="#g6-prepchroma.REF.1">1</a>). Several candidates have been discovered by the approach for cancers of the liver (<a class="bk_pop" href="#g6-prepchroma.REF.2">2</a>), the lung (<a class="bk_pop" href="#g6-prepchroma.REF.3">3</a>, <a class="bk_pop" href="#g6-prepchroma.REF.4">4</a>), the ovary (<a class="bk_pop" href="#g6-prepchroma.REF.5">5</a>), and so on.</p></dd><dt>2.</dt><dd><p class="no_top_margin">All lectins are not applicable to the affinity capture for this purpose, since there are lectins that do not have enough binding affinity for capturing glycopeptides via monovalent interaction. Lectin array analysis is commonly used for proteins as analytes, so the binding may only be detectable by the multivalent interaction between lectins and glycoproteins. Therefore, such targets may not be found by lectin affinity capture using peptides as a sample.</p></dd><dt>3.</dt><dd><p class="no_top_margin">The lectin exemplified in this protocol, AAL, has the affinity for fucosylated glycans containing, e.g., Fucα1-6GlcNAc (at chitobiose core of <i>N</i>-glycan), Fucα1-2Gal, Fucα1-3GlcNAc, and Fucα1-4GlcNAc (<a class="bk_pop" href="#g6-prepchroma.REF.6">6</a>).</p></dd></dl></div></div><div id="g6-prepchroma.References"><h2 id="_g6-prepchroma_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g6-prepchroma.REF.1">Narimatsu H, Sawaki H, Kuno A, Kaji H, Ito H, Ikehara Y. A strategy for discovery of cancer glyco-biomarkers in serum using newly developed technologies for glycoproteomics. <span><span class="ref-journal">FEBS J. </span>2010 Jan;<span class="ref-vol">277</span>(1):95–105.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19919546" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19919546</span></a>] [<a href="http://dx.crossref.org/10.1111/j.1742-4658.2009.07430.x" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g6-prepchroma.REF.2">Kaji H, Ocho M, Togayachi A, Kuno A, Sogabe M, Ohkura T, Nozaki H, Angata T, Chiba Y, Ozaki H, Hirabayashi J, Tanaka Y, Mizokami M, Ikehara Y, Narimatsu H. Glycoproteomic discovery of serological biomarker candidates for HCV/HBV infection-associated liver fibrosis and hepatocellular carcinoma. <span><span class="ref-journal">J Proteome Res. </span>2013 Jun 7;<span class="ref-vol">12</span>(6):2630–40.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/23586699" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23586699</span></a>] [<a href="http://dx.crossref.org/10.1021/pr301217b" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g6-prepchroma.REF.3">Hirao Y, Matsuzaki H, Iwaki J, Kuno A, Kaji H, Ohkura T, Togayachi A, Abe M, Nomura M, Noguchi M, Ikehara Y, Narimatsu H. Glycoproteomics approach for identifying Glycobiomarker candidate molecules for tissue type classification of non-small cell lung carcinoma. <span><span class="ref-journal">J Proteome Res. </span>2014 Nov 7;<span class="ref-vol">13</span>(11):4705–16.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/25244057" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 25244057</span></a>] [<a href="http://dx.crossref.org/10.1021/pr5006668" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g6-prepchroma.REF.4">Togayachi A, Iwaki J, Kaji H, Matsuzaki H, Kuno A, Hirao Y, Nomura M, Noguchi M, Ikehara Y, Narimatsu H. Glycobiomarker, fucosylated short-form secretogranin III levels are increased in serum of patients with small cell lung carcinoma. <span><span class="ref-journal">J Proteome Res. </span>2017 Dec 1;<span class="ref-vol">16</span>(12):4495–4505.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/28949141" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 28949141</span></a>] [<a href="http://dx.crossref.org/10.1021/acs.jproteome.7b00484" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g6-prepchroma.REF.5">Sogabe M, Nozaki H, Tanaka N, Kubota T, Kaji H, Kuno A, Togayachi A, Gotoh M, Nakanishi H, Nakanishi T, Mikami M, Suzuki N, Kiguchi K, Ikehara Y, Narimatsu H. Novel glycobiomarker for ovarian cancer that detects clear cell carcinoma. <span><span class="ref-journal">J Proteome Res. </span>2014 Mar 7;<span class="ref-vol">13</span>(3):1624–35.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/24498956" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24498956</span></a>] [<a href="http://dx.crossref.org/10.1021/pr401109n" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g6-prepchroma.REF.6">Wimmerova M, Mitchell E, Sanchez JF, Gautier C, Imberty A. Crystal structure of fungal lectin: six-bladed beta-propeller fold and novel fucose recognition mode for Aleuria aurantia lectin. <span><span class="ref-journal">J Biol Chem. </span>2003 Jul 18;<span class="ref-vol">278</span>(29):27059–67.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12732625" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12732625</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M302642200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593913_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g6-prepchroma.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="g6-prepchroma.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593913/bin/g6-prepchroma-Image001.jpg" alt="Figure 1: . Schematic presentation of chromatogram of glycopeptide capturing using lectin affinity chromatography (e." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Schematic presentation of chromatogram of glycopeptide capturing using lectin affinity chromatography (e.g., AAL). The column (e.g., 5 mm id, 50 mm: bed volume: ~1 mL) is equilibrated with a buffer (0.1 mL/min). To wash the column, the elution buffer containing elution sugar (20 mM of Fucose for AAL) is injected using a manual injector (loop size: 5 mL) twice. After injecting the sample and collection of pass fraction, glycopeptides are eluted with 20 mM of Fucose/buffer, and the bound fraction is recovered. To remove the elution sugar, glycopeptide fraction is applied to reversed-phase chromatography (C18 column: e.g., Mightysil RP-18GP).</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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