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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="Multidimensional high-performance liquid chromatography-mapping method for N-linked glycan analysis" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2022/03/14" /><meta name="citation_author" content="Hirokazu Yagi" /><meta name="citation_author" content="Koichi Kato" /><meta name="citation_pmid" content="37590684" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK593948/" /><meta name="citation_keywords" content="high-performance liquid chromatography (HPLC) map" /><meta name="citation_keywords" content="N-glycan" /><meta name="citation_keywords" content="ODS column" /><meta name="citation_keywords" content="amide column" /><meta name="citation_keywords" content="DEAE column" /><meta name="citation_keywords" content="GALAXY" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Multidimensional high-performance liquid chromatography-mapping method for N-linked glycan analysis" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Hirokazu Yagi" /><meta name="DC.Contributor" content="Koichi Kato" /><meta name="DC.Date" content="2022/03/14" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK593948/" /><meta name="description" content="Multidimensional high-performance liquid chromatography (HPLC) mapping is an effective method for determining N-glycan structures at the molecular, cellular, and tissue levels (1–6). In this method, the N-glycans are liberated from glycoproteins and labeled with 2-aminopyridine. The pyridylaminated (PA)-glycans were sequentially separated by three kinds of HPLC columns. The structures are identified on the basis of their elution positions on these HPLC columns by direct comparison with the HPLC data of available reference compounds. HPLC data from ~600 different N-glycans containing neutral, sialylated, sulfated, and glucuronylated oligosaccharides have been accumulated (7–10) and are currently available in a web application, GALAXY (11,12)." /><meta name="og:title" content="Multidimensional high-performance liquid chromatography-mapping method for N-linked glycan analysis" /><meta name="og:type" content="book" /><meta name="og:description" content="Multidimensional high-performance liquid chromatography (HPLC) mapping is an effective method for determining N-glycan structures at the molecular, cellular, and tissue levels (1–6). In this method, the N-glycans are liberated from glycoproteins and labeled with 2-aminopyridine. The pyridylaminated (PA)-glycans were sequentially separated by three kinds of HPLC columns. The structures are identified on the basis of their elution positions on these HPLC columns by direct comparison with the HPLC data of available reference compounds. HPLC data from ~600 different N-glycans containing neutral, sialylated, sulfated, and glucuronylated oligosaccharides have been accumulated (7–10) and are currently available in a web application, GALAXY (11,12)." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK593948/" /><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/g156-mappingN-linked/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK593948/" /><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="_NBK593948_"><span class="title" itemprop="name">Multidimensional high-performance liquid chromatography-mapping method for <i>N</i>-linked glycan analysis</span></h1><div class="contrib half_rhythm"><span itemprop="author">Hirokazu Yagi</span>, Doctor of Pharmaceutical sciences, Ph.D.<div class="affiliation small">Graduate School of Pharmaceutical Sciences, Nagoya City University and Exploratory Research Center on Life and Living Systems (ExCELLS), National Institute of Natural Sciences (NINS)<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.uc-ayogan.rahp@igayh" class="oemail">pj.ca.uc-ayogan.rahp@igayh</a></div></div><div class="small">Corresponding author.</div></div><div class="contrib half_rhythm"><span itemprop="author">Koichi Kato</span>, Doctor of Pharmaceutical sciences, Ph.D.<div class="affiliation small">Graduate School of Pharmaceutical Sciences, Nagoya City University and Exploratory Research Center on Life and Living Systems (ExCELLS), National Institute of Natural Sciences (NINS)<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.uc-ayogan.rahp@otakk" class="oemail">pj.ca.uc-ayogan.rahp@otakk</a></div></div></div><p class="small">Created: <span itemprop="datePublished">January 10, 2022</span>; Last Revision: <span itemprop="dateModified">March 14, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g156-mappingN-linked.Introduction"><h2 id="_g156-mappingN-linked_Introduction_">Introduction</h2><p>Multidimensional high-performance liquid chromatography (HPLC) mapping is an effective method for determining <i>N</i>-glycan structures at the molecular, cellular, and tissue levels (<a class="bk_pop" href="#g156-mappingN-linked.REF.1">1</a>–<a class="bk_pop" href="#g156-mappingN-linked.REF.6">6</a>). In this method, the <i>N</i>-glycans are liberated from glycoproteins and labeled with 2-aminopyridine. The pyridylaminated (PA)-glycans were sequentially separated by three kinds of HPLC columns. The structures are identified on the basis of their elution positions on these HPLC columns by direct comparison with the HPLC data of available reference compounds. HPLC data from ~600 different <i>N</i>-glycans containing neutral, sialylated, sulfated, and glucuronylated oligosaccharides have been accumulated (<a class="bk_pop" href="#g156-mappingN-linked.REF.7">7</a>–<a class="bk_pop" href="#g156-mappingN-linked.REF.10">10</a>) and are currently available in a web application, GALAXY (<a class="bk_pop" href="#g156-mappingN-linked.REF.11">11</a>,<a class="bk_pop" href="#g156-mappingN-linked.REF.12">12</a>).</p></div><div id="g156-mappingN-linked.Protocol"><h2 id="_g156-mappingN-linked_Protocol_">Protocol</h2><p>This section describes the structural analysis of <i>N</i>-glycans derived from cells as starting materials.</p><div id="g156-mappingN-linked.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Ultrapure water</p></dd><dt>2.</dt><dd><p class="no_top_margin">Lysis buffer: 25 mM Tris-HCl buffer (pH 7.6), 150 mM NaCl, 1 mM Ethylenediaminetetraacetic acid (EDTA), and 1% Triton X</p></dd><dt>3.</dt><dd><p class="no_top_margin">A 26-gauge syringe needle</p></dd><dt>4.</dt><dd><p class="no_top_margin">Hydrazine anhydrite, purity > 99%</p></dd><dt>5.</dt><dd><p class="no_top_margin">Screw test glass tube with screw cap (TST-SCR16-100, Iwaki, Shizuoka, Japan)</p></dd><dt>6.</dt><dd><p class="no_top_margin">Acetic anhydride, purity > 99%</p></dd><dt>7.</dt><dd><p class="no_top_margin">Acetic acids, HPLC grade</p></dd><dt>8.</dt><dd><p class="no_top_margin">Dimethylamine-borane, purity > 99%</p></dd><dt>9.</dt><dd><p class="no_top_margin">Acetonitrile, HPLC grade</p></dd><dt>10.</dt><dd><p class="no_top_margin">Carbon column buffer A: 50 mM ammonium acetate buffer (pH 7)</p></dd><dt>11.</dt><dd><p class="no_top_margin">Carbon column buffer B: 50 mM triethylamine acetate buffer (pH 7)/acetonitrile (40:60)</p></dd><dt>12.</dt><dd><p class="no_top_margin">Graphite carbon column (GL-Pak Carbograph 150 mg/3 mL, GL Sciences, Tokyo, Japan)</p></dd><dt>13.</dt><dd><p class="no_top_margin">Microcrystalline cellulose for chromatography was purchased from Merck Millipore, Burlington, MA (Catalog number: 1.02331.0500)</p></dd><dt>14.</dt><dd><p class="no_top_margin">2-Aminopyridine, purity > 99%</p></dd><dt>15.</dt><dd><p class="no_top_margin">Cellulose column buffer I: 66% of 1-butanol, 16% of ethanol, and 16% of 0.6 M acetic acid</p></dd><dt>16.</dt><dd><p class="no_top_margin">Cellulose column buffer II: 33% of ethanol and 66% of 75 mM ammonium bicarbonate</p></dd><dt>17.</dt><dd><p class="no_top_margin">TSKgel diethylaminoethyl (DEAE)-5PW column (7.5-mm i.d. × 75 mm; Tosoh, Tokyo, Japan)</p></dd><dt>18.</dt><dd><p class="no_top_margin">Shim-pack HRC-octadecyl silica (ODS) column (6.0-mm i.d. × 150 mm; Shimadzu, Kyoto, Japan)</p></dd><dt>19.</dt><dd><p class="no_top_margin">TSKgel Amide-80 column (4.6-mm i.d. × 250 mm; Tosoh)</p></dd><dt>20.</dt><dd><p class="no_top_margin">PA-glucose oligomer (DP = 3–22) (Takara bio. Inc., Kusatsu, Japan)</p></dd></dl></div><div id="g156-mappingN-linked.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Heat block</p></dd><dt>2.</dt><dd><p class="no_top_margin">Water bass</p></dd><dt>3.</dt><dd><p class="no_top_margin">HPLC system with a fluorescence detector and pump for two-liquid gradient</p></dd><dt>4.</dt><dd><p class="no_top_margin">SpeedVac vacuum concentrator (Thermo Fisher Scientific, Waltham, MA)</p></dd></dl></div><div id="g156-mappingN-linked.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Prepare oligosaccharide samples from glycoproteins</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Prepare the cell lysate by adding a lysis buffer and passing the sample through a 26-gauge syringe needle (<b>Note 1</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Add four volumes of ethanol to one volume of cell or tissue lysate and then incubate at −20°C overnight for precipitation of proteins. The precipitant is dried by a rotary vacuum evaporator.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add 200 µL of hydrazine anhydride to protein fraction (~1 mg) in a 5-mL glass tube and then incubate 95°C for 10 h in a heat block or water bass (<b>Notes 2</b> and <b>3</b>).</p></dd><dt>d.</dt><dd><p class="no_top_margin">Mix the hydrazine solution with 3 mL of carbon column buffer A for the quenching reaction and load it onto a graphite carbon column.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Equilibrate a graphite carbon column with 15 mL of buffer A after the follow-through of 5 mL of buffer B.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Add the oligosaccharide sample to the column and wash with 5 mL of buffer A twice.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Finally, elute the oligosaccharide with 5 mL of buffer B containing 2% acetic anhydride into the glass tube. The eluted solution is dried by a rotary vacuum evaporator, and N-acetylated oligosaccharides were obtained in a glass tube.</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Fluorescent labeling with 2-aminopyridine (<b>Note 4</b>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">To prepare the labeling solution, dissolve 276 mg of 2-aminopyridine in 100 µL of acetic acid.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Add 30 µL of the labeling solution to the evaporated oligosaccharide sample in a separate screw test glass tube with screw cap and place in the heat block at 90°C for 60 min, with the tube tightly capped.</p></dd><dt>c.</dt><dd><p class="no_top_margin">To prepare a reducing solution, mix 200 mg of dimethylamine-borane, 50 µL of acetic acid, and 80 µL of ultrapure water.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Add 110 µL of the reducing solution in the sample tube and incubate in the heat block at 80°C for 35 min, with the tube tightly capped.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Wash microcrystalline cellulose with water eight times and then washed twice with cellulose column buffer I.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Add 2 mL of microcrystalline cellulose suspension (50%) in the cellulose column buffer I to an open column and let the solution fall through.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Wash the microcrystalline cellulose with the cellulose column buffer I twice.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Mix the pyridylaminated samples with the cocktail comprising 350 µL of ultrapure water, 400 µL of ethanol, 1600 µL of 1-butanol, and 15 µL of acetic acid.</p></dd><dt>i.</dt><dd><p class="no_top_margin">Add the mixture to the column filled with microcrystalline cellulose.</p></dd><dt>j.</dt><dd><p class="no_top_margin">After passing through the sample, the column is washed twice with 3 mL of cellulose column I.</p></dd><dt>k.</dt><dd><p class="no_top_margin">Elute PA-glycans with 2 mL of cellulose column buffer II.</p></dd><dt>l.</dt><dd><p class="no_top_margin">Evaporate the eluted fraction with a SpeedVac vacuum concentrator in order to apply the PA-glycans to HPLC columns.</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Glycan separation by a DEAE column with anion-exchange chromatography</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Running condition</p></dd><dt>b.</dt><dd><p class="no_top_margin">Column: DEAE-5PW column</p></dd><dt>c.</dt><dd><p class="no_top_margin">Column temperature: 30°C</p></dd><dt>d.</dt><dd><p class="no_top_margin">DEAE solvent A: 10% acetonitrile/0.01% triethylamine</p></dd><dt>e.</dt><dd><p class="no_top_margin">DEAE solvent B: 10% acetonitrile/7.4% triethylamine/3% acetic acid</p></dd><dt>f.</dt><dd><p class="no_top_margin">The gradient elution parameters are 5–40 min and a linear gradient of 0%–20% solvent B</p></dd><dt>g.</dt><dd><p class="no_top_margin">Flow rate: 1 mL/min</p></dd><dt>h.</dt><dd><p class="no_top_margin">Detection: Fluorescence (Ex. 320 nm, Em. 400 nm)</p></dd><dt>i.</dt><dd><p class="no_top_margin">Inject PA-glycans into the DEAE column for separation according to the contents of sialic acid residues or sulfate groups. The peaks are fractionated (<b>Note 5</b>).</p></dd><dt>j.</dt><dd><p class="no_top_margin">Evaporate the eluted fraction with a SpeedVac vacuum concentrator.</p></dd></dl></dd><dt>4.</dt><dd><p class="no_top_margin">Glycan separation by an ODS column with anion-exchange chromatography</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Running condition</p></dd><dt>b.</dt><dd><p class="no_top_margin">Column: Shim-pack HRC-ODS (6.0 × 150 mm).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Column temperature: 55°C</p></dd><dt>d.</dt><dd><p class="no_top_margin">ODS solvent A: 10 mM sodium phosphate (pH 3.8).</p></dd><dt>e.</dt><dd><p class="no_top_margin">ODS solvent B: 10 mM sodium phosphate (pH 3.8)/0.5% 1-butanol.</p></dd><dt>f.</dt><dd><p class="no_top_margin">The gradient elution parameters: 0–60 min and a linear gradient of 20%–50% of solvent B.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Flow rate: 1 mL/min</p></dd><dt>h.</dt><dd><p class="no_top_margin">Detection: Fluorescence (Ex. 320 nm, Em. 400 nm)</p></dd><dt>i.</dt><dd><p class="no_top_margin">Inject the PA-glucose oligomers into the ODS column, and record the elution time for normalization of the elution time to glucose units (GUs).</p></dd><dt>j.</dt><dd><p class="no_top_margin">Inject each fraction separated from the DEAE column.</p></dd><dt>k.</dt><dd><p class="no_top_margin">Fractionate the PA-glycans and record their elution time (<b>Note 5</b>).</p></dd><dt>l.</dt><dd><p class="no_top_margin">Evaporate the eluted fraction with a SpeedVac vacuum concentrator.</p></dd></dl></dd><dt>5.</dt><dd><p class="no_top_margin">Glycan separation by an amide column with normal-phase chromatography</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Running condition</p></dd><dt>b.</dt><dd><p class="no_top_margin">Column: TSKgel Amide-80 (4.5 × 250 mm).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Column temperature: 40°C</p></dd><dt>d.</dt><dd><p class="no_top_margin">Amide solvent A: 65% acetonitrile/2.9% triethylamine/0.6% acetic acid.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Amide solvent B: 50% acetonitrile/4.1% triethylamine/1.7% acetic acid.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Gradient elution parameters: 0–30 min with a linear gradient of 0%–60% solvent B.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Flow rate: 1 mL/min</p></dd><dt>h.</dt><dd><p class="no_top_margin">Detection: Fluorescence (Ex. 320 nm and Em. 400 nm)</p></dd><dt>i.</dt><dd><p class="no_top_margin">Inject PA-glucose oligomers, and record the elution time as GU.</p></dd><dt>j.</dt><dd><p class="no_top_margin">Inject each fraction separated from the ODS column after dissolving in amide solvent A. Each peak is fractionated and the elution time is recorded (<b>Notes 5</b> and <b>6</b>).</p></dd></dl></dd><dt>6.</dt><dd><p class="no_top_margin">Identification of the glycan structure</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Convert the elution time of the PA-glycans into GU on the ODS column and amide column (<a class="figpopup" href="/books/NBK593948/figure/g156-mappingN-linked.F1/?report=objectonly" target="object" rid-figpopup="figg156mappingNlinkedF1" rid-ob="figobg156mappingNlinkedF1">Figure 1</a>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Plot the GU on the ODS column, GU on the amide column, and the components of negatively charged residues of induvial glycans on the X-axis, Y-axis, and Z-axis, respectively.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Compare the GU value with that of the PA-glycans in the GALAXY (<a href="http://www.glycoanalysis.info/" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://www<wbr style="display:inline-block"></wbr>.glycoanalysis.info/</a>) database (<a class="bk_pop" href="#g156-mappingN-linked.REF.11">11</a>) to estimate the glycan structures. (<b>Note 6</b>).</p></dd><dt>d.</dt><dd><p class="no_top_margin">Identify the glycan structure by co-injection with standard PA-glycans.</p></dd><dt>e.</dt><dd><p class="no_top_margin">For glycans whose data do not match the known data, the glycan structure that matches the known data after the treatment of glycosidases with known specificity is considered, and the original glycan structures are estimated (<b>Note 7</b>).</p></dd></dl></dd></dl></div><div id="g156-mappingN-linked.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Glycoproteins should be desalted and dried in a centrifugal concentrator. Acetone powder from treated cells and tissues can also be used as starting materials.</p></dd><dt>2.</dt><dd><p class="no_top_margin">For better hydrazinolysis, remove water to the maximum possible extent using desiccation or evaporation.</p></dd><dt>3.</dt><dd><p class="no_top_margin">Instead of hydrazinolysis, glycans can be released by glycoamidease A or peptide:<i>N</i>-glycosidase F (PNGaseF) treatment.</p></dd><dt>4.</dt><dd><p class="no_top_margin">About 10% of epimerized material is obtained as a byproduct of the pyridylamination reaction.</p></dd><dt>5.</dt><dd><p class="no_top_margin">Dissolve the dried sample in 20 µL of solvent A, and first check the elution profile by passing 1 µL through the HPLC column. Then, divide the remaining sample into several fractions.</p></dd><dt>6.</dt><dd><p class="no_top_margin">The predicted structure in the GALAXY database can be examined using the mass value obtained by applying mass spectrometry to each fraction after the fractionation of the ODS column (<a class="bk_pop" href="#g156-mappingN-linked.REF.11">11</a>,<a class="bk_pop" href="#g156-mappingN-linked.REF.12">12</a>).</p></dd><dt>7.</dt><dd><p class="no_top_margin">The elution time of PA-glycans from ODS or amide columns can be regarded as the sum of the affinity (unit contribution [UC]) of the component monosaccharide residues to the columns (<a class="bk_pop" href="#g156-mappingN-linked.REF.13">13</a>). The UC value of each sugar residue is calculated using multiple regression analysis. With this UC value, it is possible to calculate the GU value of those PA-glycans for which no data are available on the map.</p></dd></dl></div></div><div id="g156-mappingN-linked.References"><h2 id="_g156-mappingN-linked_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.1">Yagi H, Saito T, Yanagisawa M, Yu RK, Kato K. Lewis X-carrying N-Glycans Regulate the Proliferation of Mouse Embryonic Neural Stem Cells via the Notch Signaling Pathway. <span><span class="ref-journal">J Biol Chem. </span>2012 Jul 13;<span class="ref-vol">287</span>(29):24356–64.</span> [<a href="/pmc/articles/PMC3397862/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3397862</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22645129" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22645129</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M112.365643" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.2">Yagi H, Watanabe S, Suzuki T, Takahashi T, Suzuki Y, Kato K. Comparative analyses of N-Glycosylation profiles of influenza a viruses grown in different host cells. <span><span class="ref-journal">Open Glycosci. </span>2012;<span class="ref-vol">5</span>(1):2–12.</span> [<a href="http://dx.crossref.org/10.2174/1875398101205010002" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.3">Yagi H, Yamamoto M, Yu S-Y, Takahashi N, Khoo K-H, Lee YC, et al. N-Glycosylation profiling of turtle egg yolk: expression of galabiose structure. <span><span class="ref-journal">Carbohydr Res. </span>2010 Feb 11;<span class="ref-vol">345</span>(3):442–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/20044081" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20044081</span></a>] [<a href="http://dx.crossref.org/10.1016/j.carres.2009.12.002" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.4">Thongratsakul S, Songserm T, Poolkhet C, Kondo S, Yagi H, Hiramatsu H, et al. Determination of N-Linked Sialyl-Sugar Chains in the Lungs of Domestic Cats and Dogs in Thailand Susceptible to the Highly Pathogenic Avian Influenza Virus (H5N1). <span><span class="ref-journal">Open Glycosci. </span>2009 Aug;<span class="ref-vol">2</span>(1):28–36.</span> [<a href="http://dx.crossref.org/10.2174/1875398100902010028" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.5">Yagi H, Yasukawa N, Yu S-Y, Guo C-T, Takahashi N, Takahashi T, et al. The expression of sialylated high-antennary N-glycans in edible bird’s nest. <span><span class="ref-journal">Carbohydr Res. </span>2008 Jun 9;<span class="ref-vol">343</span>(8):1373–7.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18439991" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18439991</span></a>] [<a href="http://dx.crossref.org/10.1016/j.carres.2008.03.031" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.6">Yagi H, Takahashi N, Yamaguchi Y, Kato K. Temperature-dependent isologous Fab-Fab interaction that mediates cryocrystallization of a monoclonal immunoglobulin G. <span><span class="ref-journal">Mol Immunol. </span>2004 Nov;<span class="ref-vol">41</span>(12):1211–5.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15482856" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15482856</span></a>] [<a href="http://dx.crossref.org/10.1016/j.molimm.2004.06.003" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.7">Nakagawa H, Kawamura Y, Kato K, Shimada I, Arata Y, Takahashi N. Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography. <span><span class="ref-journal">Anal Biochem. </span>1995 Mar 20;<span class="ref-vol">226</span>(1):130–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7785764" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7785764</span></a>] [<a href="http://dx.crossref.org/10.1006/abio.1995.1200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.8">Tomiya N, Awaya J, Kurono M, Endo S, Arata Y, Takahashi N. Analyses of N-linked oligosaccharides using a two-dimensional mapping technique. <span><span class="ref-journal">Anal Biochem. </span>1988 May 15;<span class="ref-vol">171</span>(1):73–90.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/3407923" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 3407923</span></a>] [<a href="http://dx.crossref.org/10.1016/0003-2697(88)90126-1" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.9">Yagi H, Takahashi N, Yamaguchi Y, Kimura N, Uchimura K, Kannagi R, et al. Development of structural analysis of sulfated N-glycans by multidimensional high performance liquid chromatography mapping methods. <span><span class="ref-journal">Glycobiology. </span>2005 Oct;<span class="ref-vol">15</span>(10):1051–60.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15958418" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15958418</span></a>] [<a href="http://dx.crossref.org/10.1093/glycob/cwi092" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.10">Yagi H, Yamada K, Ohno E, Utsumi M, Yamaguchi Y, Kurimoto E, et al. Development and Application of High Performance Liquid Chromatography Map of Glucuronyl N-glycans. <span><span class="ref-journal">Open Glycosci. </span>2008 Jun;<span class="ref-vol">1</span>(1):8–18.</span> [<a href="http://dx.crossref.org/10.2174/1875398100801010008" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.11">Takahashi N, Kato K. GALAXY(Glycoanalysis by the three axes of MS and chromatography): A web application that assists structural analyses of N-glycans. <span><span class="ref-journal">Trends Glycosci Glycotechnol. </span>2003 Jul 2;<span class="ref-vol">15</span>(84):235–51.</span> doi: . [<a href="http://dx.crossref.org/10.4052/tigg.15.235" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.12">Yagi H, Amagasa E, Shiota M, Yamada I, Aoki-Kinoshita KF, Kato K. GALAXY ver3: updated web application for glycosylation profiling based on 3D HPLC map. <span><span class="ref-journal">Glycobiology. </span>2022 Jul 13;<span class="ref-vol">32</span>(8):646–650.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/35452093" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 35452093</span></a>] [<a href="http://dx.crossref.org/10.1093/glycob/cwac025" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="g156-mappingN-linked.REF.13">Tomiya N, Takahashi N. Contribution of component monosaccharides to the coordinates of neutral and sialyl pyridylaminated N-glycans on a two-dimensional sugar map. <span><span class="ref-journal">Anal Biochem. </span>1998 Nov 15;<span class="ref-vol">264</span>(2):204–10.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9866684" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9866684</span></a>] [<a href="http://dx.crossref.org/10.1006/abio.1998.2849" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593948_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g156-mappingN-linked.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="g156-mappingN-linked.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593948/bin/g156-mappingN-linked-Image001.jpg" alt="Figure 1: . Elution profiles of the standard PA-glucose oligomers and sample PA-glycan in the octadecyl-silica (ODS) column." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Elution profiles of the standard PA-glucose oligomers and sample PA-glycan in the octadecyl-silica (ODS) column.</p><p>The dotted line shows glucose oligomers (degree of polymerization 4–21), and the solid line shows a sample <i>N</i>-glycan. The sample glycans elute between 12 and 13 oligomers, and the elution time can be converted to 12.5 GU by proportional distribution.</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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