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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="N-Glycan profiling by liquid chromatography-mass spectrometry (LC-MS)" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2021/12/21" /><meta name="citation_author" content="Miyako Nakano" /><meta name="citation_pmid" content="37590738" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK594013/" /><meta name="citation_keywords" content="N-glycan" /><meta name="citation_keywords" content="LC-MS" /><meta name="citation_keywords" content="aminoxyTMT™ (Tandem Mass Tag™)" /><meta name="citation_keywords" content="alditol" /><meta name="citation_keywords" content="structural analysis" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="N-Glycan profiling by liquid chromatography-mass spectrometry (LC-MS)" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Miyako Nakano" /><meta name="DC.Date" content="2021/12/21" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK594013/" /><meta name="description" content="For N-glycan profiling using liquid chromatography-mass spectrometry (LC-MS), derivatization of N-glycans is required to analyze structures of N-glycans released from glycoproteins with more sensitive and simpler detection. There are two reasons. First, MS can only detect ionic molecules with a positive or negative charge, but N-glycans have no charge except for sialic acid. Second, N-glycans released by PNGaseF (also known as peptide-N-glycosidaseF or N-glycanase) have α-anomer and β-anomer in the reducing end, which is complicated because the two peaks are observed in one structure. Thus, we analyzed the N-glycans using LC-MS after derivatization to give the released N-glycans an electric charge or to eliminate the anomeric form. Various derivatization methods for N-glycans have been known so far (1), but this chapter introduces two derivatization methods for N-glycan profiling (alditol reaction and aminoxyTMT™ (Tandem Mass Tag™) labeling)." /><meta name="og:title" content="N-Glycan profiling by liquid chromatography-mass spectrometry (LC-MS)" /><meta name="og:type" content="book" /><meta name="og:description" content="For N-glycan profiling using liquid chromatography-mass spectrometry (LC-MS), derivatization of N-glycans is required to analyze structures of N-glycans released from glycoproteins with more sensitive and simpler detection. There are two reasons. First, MS can only detect ionic molecules with a positive or negative charge, but N-glycans have no charge except for sialic acid. Second, N-glycans released by PNGaseF (also known as peptide-N-glycosidaseF or N-glycanase) have α-anomer and β-anomer in the reducing end, which is complicated because the two peaks are observed in one structure. Thus, we analyzed the N-glycans using LC-MS after derivatization to give the released N-glycans an electric charge or to eliminate the anomeric form. Various derivatization methods for N-glycans have been known so far (1), but this chapter introduces two derivatization methods for N-glycan profiling (alditol reaction and aminoxyTMT™ (Tandem Mass Tag™) labeling)." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK594013/" /><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/g134-NglycanLCMS/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK594013/" /><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="_NBK594013_"><span class="title" itemprop="name"><i>N</i>-Glycan profiling by liquid chromatography-mass spectrometry (LC-MS)</span></h1><div class="contrib half_rhythm"><span itemprop="author">Miyako Nakano</span>, Dr.<div class="affiliation small">Hiroshima University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-amihsorih@onakanim" class="oemail">pj.ca.u-amihsorih@onakanim</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">October 24, 2021</span>; Last Update: <span itemprop="dateModified">December 21, 2021</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g134-NglycanLCMS.Introduction"><h2 id="_g134-NglycanLCMS_Introduction_">Introduction</h2><p>For <i>N</i>-glycan profiling using liquid chromatography-mass spectrometry (LC-MS), derivatization of <i>N</i>-glycans is required to analyze structures of <i>N</i>-glycans released from glycoproteins with more sensitive and simpler detection. There are two reasons. First, MS can only detect ionic molecules with a positive or negative charge, but <i>N</i>-glycans have no charge except for sialic acid. Second, <i>N</i>-glycans released by PNGaseF (also known as peptide-<i>N</i>-glycosidaseF or <i>N</i>-glycanase) have α-anomer and β-anomer in the reducing end, which is complicated because the two peaks are observed in one structure. Thus, we analyzed the <i>N</i>-glycans using LC-MS after derivatization to give the released <i>N</i>-glycans an electric charge or to eliminate the anomeric form. Various derivatization methods for <i>N</i>-glycans have been known so far (<a class="bk_pop" href="#g134-NglycanLCMS.REF.1">1</a>), but this chapter introduces two derivatization methods for <i>N</i>-glycan profiling (alditol reaction and aminoxyTMT™ (Tandem Mass Tag™) labeling).</p></div><div id="g134-NglycanLCMS.Protocol"><h2 id="_g134-NglycanLCMS_Protocol_">Protocol</h2><p>In this chapter, first, releasing method 1) of <i>N</i>-glycans from glycoproteins to conduct glycomic analysis with comprehensive and routine sample preparation will be described, and next, two kinds of derivatization methods will be described for <i>N</i>-glycan structural analysis using LC-MS; 2) that using the alditol reaction; and 3) that using aminoxyTMT labeling of released <i>N</i>-glycans using glycoprotein model “bovine fetuin,” which has a well-known structure and relative abundance of <i>N</i>-glycan (<a class="bk_pop" href="#g134-NglycanLCMS.REF.2">2</a>).</p><div id="g134-NglycanLCMS.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">For the preparation of released <i>N</i>-glycans</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Bovine fetuin (from bovine serum) as a glycoprotein standard.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Polyvinylidene difluoride (PVDF) membrane.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) device and gel (in case of blot).</p></dd><dt>d.</dt><dd><p class="no_top_margin">Direct Blue 71 (tetrasodium;3-[[4-[[4-[(6-amino-1-hydroxy-3-sulfonatonaphthalen-2-yl)diazenyl]-6-sulfonatonaphthalen-1-yl]diazenyl]naphthalen-1-yl]diazenyl]naphthalene-1,5-disulfonate)</p></dd><dt>e.</dt><dd><p class="no_top_margin">Polyvinylpyrrolidone 40000</p></dd><dt>f.</dt><dd><p class="no_top_margin">PNGaseF (recombinant form of the gene from <i>Flavobacterium meningosepticum</i>, Roche)</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">For alditol reaction of released <i>N</i>-glycans</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Sodium borohydride, NaBH4.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Cation exchange resin (Dowex 50W × 8, 200–400 mesh H<sup>+</sup> form).</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">For aminoxyTMT labeling of released <i>N</i>-glycans</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">aminoxyTMTsixplex Label Reagent Set (aminoxyTMT6-126, 127, 128, 129, 130, and 131, Thermo Fisher Scientific).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Sepharose CL4B.</p></dd></dl></dd></dl></div><div id="g134-NglycanLCMS.Instruments"><h3>Instruments</h3><p>1. Vacuum-Centrifugal Concentrator (Tomy Seiko).</p><p>2. Vanquish HPLC system (Thermo Fisher Scientific).</p><p>3. LTQ Orbitrap XL (Thermo Fisher Scientific) with ESI source (Thermo Fisher Scientific).</p></div><div id="g134-NglycanLCMS.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Protocol for preparation of released <i>N</i>-glycans (<a class="bk_pop" href="#g134-NglycanLCMS.REF.3">3</a>–<a class="bk_pop" href="#g134-NglycanLCMS.REF.5">5</a>) (<a class="figpopup" href="/books/NBK594013/figure/g134-NglycanLCMS.F1/?report=objectonly" target="object" rid-figpopup="figg134NglycanLCMSF1" rid-ob="figobg134NglycanLCMSF1">Figure 1</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Dissolve glycoprotein(s) in water or 8 M urea (bovine fetuin: 4 mg/mL) (<b>Note 1</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">SDS-PAGE after boiling glycoprotein(s) sample with a reducing agent (bovine fetuin: 10 μg/lane).</p></dd><dt>i.</dt><dd><p class="no_top_margin">Blot (=Transfer) separated glycoprotein(s) onto PVDF membrane (bovine fetuin: 10 μg/band) (<a class="bk_pop" href="#g134-NglycanLCMS.REF.6">6</a>,<a class="bk_pop" href="#g134-NglycanLCMS.REF.7">7</a>), or</p></dd><dt>ii.</dt><dd><p class="no_top_margin">Dot glycoprotein(s) onto a PVDF membrane (bovine fetuin: 10 μg/dot, 2.5 μL × 4 times) (<a class="bk_pop" href="#g134-NglycanLCMS.REF.8">8</a>,<a class="bk_pop" href="#g134-NglycanLCMS.REF.9">9</a>) (<b>Note 2</b>).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Stain glycoprotein(s) on the PVDF membrane with Direct Blue 71 (Sigma-Aldrich) (800 µL solution A [0.1% Direct Blue 71] in 10 mL solution B [acetic acid:ethanol:water = 1:4:5]) for 5 min. After destaining with solution B for 1 min, the membrane was dried at room temperature for >3 h.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Cut glycoprotein band or glycoprotein dot from the PVDF membrane and place in separate wells of a 96-well microtiter plate.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Cover the band or dot with 100 µL of 1% (w/v) polyvinylpyrrolidone 40000 in 50% (v/v) methanol. Then, wash it with water, agitate for 20 min, and wash with water (100 µL × 5 times).</p></dd><dt>f.</dt><dd><p class="no_top_margin">Add PNGase F (2U in 10 µL of 20 mM phosphate buffer, pH 7.3, Roche) to each well and incubate at 37°C for 15 min, and then, add 10 μL of water to each well and incubate at 37°C overnight to release <i>N</i>-glycans.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Collect the released <i>N</i>-glycans and transfer them to 1.5 mL polypropylene tubes.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Add ammonium acetate buffer (100 mM, pH 5.0, 20 μL) to the tube and incubate it for 1 h at room temperature to completely generate the reducing terminus (<b>Note 3</b>) and then, evaporate to dryness.</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Protocol for alditol reaction of released <i>N</i>-glycans and LC-MS analysis (<a class="bk_pop" href="#g134-NglycanLCMS.REF.3">3</a>–<a class="bk_pop" href="#g134-NglycanLCMS.REF.5">5</a>,<a class="bk_pop" href="#g134-NglycanLCMS.REF.10">10</a>) (<a class="figpopup" href="/books/NBK594013/figure/g134-NglycanLCMS.F1/?report=objectonly" target="object" rid-figpopup="figg134NglycanLCMSF1" rid-ob="figobg134NglycanLCMSF1">Figures 1</a> and <a class="figpopup" href="/books/NBK594013/figure/g134-NglycanLCMS.F2/?report=objectonly" target="object" rid-figpopup="figg134NglycanLCMSF2" rid-ob="figobg134NglycanLCMSF2">2</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Add 20 μL of 1 M NaBH<sub>4</sub> in 50 mM KOH to the sample tube containing dried released <i>N</i>-glycans and reduce <i>N</i>-glycan terminus with incubation at 50°C for 3 h to generate alditol <i>N</i>-glycans.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Add 1 μL of acetic acid to stop the reduction reaction.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Apply the sample solution to the cation exchange column to desalt (<b>Notes 4</b> and <b>5</b>).</p></dd><dt>d.</dt><dd><p class="no_top_margin">Receive the pass though solution with a 1.5 mL polypropylene tube containing 200 µL of 100 mM NH<sub>4</sub>HCO<sub>3</sub>. Also, receive water (50 μL twice) to wash the column, and evaporate the solution to dryness.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Remove the remaining borate by adding (100 μL × 3) methanol and drying under vacuum.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Resuspend alditol <i>N</i>-glycans with 10 mM NH<sub>4</sub>HCO<sub>3</sub> (20 μL) before glycan analysis using LC-MS.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Analyze structures of alditol <i>N</i>-glycans by LC-MS under the following conditions:</p><ul class="simple-list"><li class="half_rhythm"><div>LC:</div><ul><li class="half_rhythm"><div>Injection volume: 8 μL</div></li><li class="half_rhythm"><div>Column: 5 μm HyperCarb, 1 mm I.D. × 100 μm (Thermo Fisher Scientific)</div></li><li class="half_rhythm"><div>Flow rate: 50 μL/min</div></li><li class="half_rhythm"><div>Column oven: 40°C</div></li><li class="half_rhythm"><div>Elute: a sequence of isocratic and two segmented linear gradients; 0–8 min, 10 mM NH<sub>4</sub>HCO<sub>3</sub>; 8–38 min, 6.75%–15.75% (v/v) acetonitrile in 10 mM NH<sub>4</sub>HCO<sub>3</sub>; 38–73 min, 15.75%–40.5% (v/v) acetonitrile in 10 mM NH<sub>4</sub>HCO<sub>3</sub>; increasing to 81% (v/v) acetonitrile in 10 mM NH<sub>4</sub>HCO<sub>3</sub> for 7 min; and re-equilibrated with 10 mM NH<sub>4</sub>HCO<sub>3</sub> for 15 min.</div></li><li class="half_rhythm"><div>The eluate was continuously introduced into an ESI source.</div></li></ul></li><li class="half_rhythm"><div>MS:</div><ul><li class="half_rhythm"><div>Polarity: negative ion mode.</div></li><li class="half_rhythm"><div>Mass range: <i>m/z</i> 500–<i>m/z</i> 2500.</div></li><li class="half_rhythm"><div>Capillary temperature: 300°C.</div></li><li class="half_rhythm"><div>Source voltage: 3.0 kV.</div></li><li class="half_rhythm"><div>Capillary voltage: −18 V.</div></li><li class="half_rhythm"><div>Tube lens voltage: −112.80 V.</div></li></ul></li><li class="half_rhythm"><div>MS/MS:</div><ul class="simple-list"><li class="half_rhythm"><div>Iontrap via collision-induced dissociation (CID) (the top three precursor ions were fragmented via CID)</div></li></ul></li></ul></dd><dt>h.</dt><dd><p class="no_top_margin">Analyze structures of alditol <i>N</i>-glycans from the LC-MS data (<a class="figpopup" href="/books/NBK594013/figure/g134-NglycanLCMS.F2/?report=objectonly" target="object" rid-figpopup="figg134NglycanLCMSF2" rid-ob="figobg134NglycanLCMSF2">Figure 2</a>).</p><p>The monoisotopic mass of each alditol <i>N</i>-glycan was assigned to a possible monosaccharide composition using the GlycoMod software tool (mass tolerance for precursor ions of ±0.006 Da, https://web.expasy.org/glycomod/), and the proposed glycan structures were further verified through retention time on the basis of a previous report (<a class="bk_pop" href="#g134-NglycanLCMS.REF.4">4</a>,<a class="bk_pop" href="#g134-NglycanLCMS.REF.10">10</a>). Xcalibur software ver. 2.2. (Thermo Fisher Scientific) was used to show base peak chromatogram (BPC) and extracted ion chromatogram (EIC) and to analyze MS and MS/MS data (<b>Note 6</b>).</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Protocol for aminoxyTMT labeling of released <i>N</i>-glycans and LC-MS analysis (<a class="bk_pop" href="#g134-NglycanLCMS.REF.7">7</a>,<a class="bk_pop" href="#g134-NglycanLCMS.REF.9">9</a>,<a class="bk_pop" href="#g134-NglycanLCMS.REF.11">11</a>) (<a class="figpopup" href="/books/NBK594013/figure/g134-NglycanLCMS.F1/?report=objectonly" target="object" rid-figpopup="figg134NglycanLCMSF1" rid-ob="figobg134NglycanLCMSF1">Figures 1</a>–<a class="figpopup" href="/books/NBK594013/figure/g134-NglycanLCMS.F3/?report=objectonly" target="object" rid-figpopup="figg134NglycanLCMSF3" rid-ob="figobg134NglycanLCMSF3">3</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Add aminoxyTMT6 reagent (0.02 mg in 200 μL of 95% methanol, 0.1% acetic acid solution) to the sample tube containing dried released <i>N</i>-glycans and react by continuous shaking for 15 min at room temperature.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Evaporate the reaction solution, add 200 µL of 95% methanol to the samples, and shake for 15 min.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Evaporate the samples, add 100 µL of 10% acetone solution to the samples, and incubate at room temperature for 15 min with continuous shaking.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Evaporate the samples.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Remove excess reagent by solid-phase extraction using Sepharose CL4B (<b>Note 7</b>).</p></dd><dt>f.</dt><dd><p class="no_top_margin">Resuspend aminoxyTMT <i>N</i>-glycans with 10 mM NH<sub>4</sub>HCO<sub>3</sub> (20 µL) before glycan analysis via LC-MS.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Analyze structures of aminoxyTMT <i>N</i>-glycans via LC-MS under the following condition.</p><ul class="simple-list"><li class="half_rhythm"><div>LC:</div><ul class="simple-list"><li class="half_rhythm"><div>Injection volume: 8 µL.</div></li><li class="half_rhythm"><div>Column: 5 µm HyperCarb, 1 mm I.D. × 100 µm.</div></li><li class="half_rhythm"><div>Flow rate: 50 µL/min.</div></li><li class="half_rhythm"><div>Column oven: 40°C.</div></li><li class="half_rhythm"><div>Elute: a sequence of isocratic and two segmented linear gradients: 0–8 min, 10 mM NH<sub>4</sub>HCO<sub>3</sub>; 8–38 min, 9%–22.5% (v/v) acetonitrile in 10 mM NH<sub>4</sub>HCO<sub>3</sub>; 38–73 min, 22.5%–51.75% (v/v) acetonitrile in 10 mM NH<sub>4</sub>HCO<sub>3</sub>; increasing to 81% (v/v) acetonitrile in 10 mM NH<sub>4</sub>HCO<sub>3</sub> for 7 min; and re-equilibrated with 10 mM NH<sub>4</sub>HCO<sub>3</sub> for 15 min.</div></li><li class="half_rhythm"><div>The eluate was continuously introduced into an ESI source.</div></li></ul></li><li class="half_rhythm"><div>MS:</div><ul class="simple-list"><li class="half_rhythm"><div>Polarity: positive ion mode.</div></li><li class="half_rhythm"><div>Mass range: <i>m/z</i> 800–<i>m/z</i> 2000.</div></li><li class="half_rhythm"><div>Capillary temperature: 300°C.</div></li><li class="half_rhythm"><div>Source voltage: 4.5 kV.</div></li><li class="half_rhythm"><div>Capillary voltage: 18 V.</div></li><li class="half_rhythm"><div>Tube lens voltage: 110 V.</div></li></ul></li><li class="half_rhythm"><div>MS/MS:</div><ul class="simple-list"><li class="half_rhythm"><div>Orbitrap by higher-energy C-trap dissociation (HCD) (the top three precursor ions were fragmented by HCD using a stepped collision energy [normalized collision energy: 35.0; the width: 40.0; the steps: 3; minimum signal required: 10000; isolation width: 4.00; and activation time: 100] using Orbitrap).</div></li></ul></li></ul></dd><dt>h.</dt><dd><p class="no_top_margin">Analyze structures of aminoxyTMT <i>N</i>-glycans from the LC-MS data (<a class="figpopup" href="/books/NBK594013/figure/g134-NglycanLCMS.F2/?report=objectonly" target="object" rid-figpopup="figg134NglycanLCMSF2" rid-ob="figobg134NglycanLCMSF2">Figures 2</a> and <a class="figpopup" href="/books/NBK594013/figure/g134-NglycanLCMS.F3/?report=objectonly" target="object" rid-figpopup="figg134NglycanLCMSF3" rid-ob="figobg134NglycanLCMSF3">3</a>).</p><p>The monoisotopic mass of each aminoxyTMT <i>N</i>-glycan was assigned to a possible monosaccharide composition using the GlycoMod software tool (mass tolerance for precursor ions of ±0.006 Da), and the proposed glycan structures were further verified through annotation using a fragmentation mass matching approach based on the MS/MS data. Xcalibur software ver. 2.2. was used to show BPC and EIC and analyze MS and MS/MS data (<a class="figpopup" href="/books/NBK594013/figure/g134-NglycanLCMS.F3/?report=objectonly" target="object" rid-figpopup="figg134NglycanLCMSF3" rid-ob="figobg134NglycanLCMSF3">Figure 3</a>) (<b>Note 8</b>).</p></dd></dl></dd></dl></div><div id="g134-NglycanLCMS.Notes"><h3>Notes</h3><p>1. Dissolve insoluble glycoproteins in 8 M urea.</p><p>2. Do not dot more than 2.5 μL/once onto PVDF. Do not exceed the size of the holes of a 96-well microtiter plate.</p><p>3. In the case of the following aminoxyTMT labeling, it is not necessary to generate the reducing terminus with a weak acid (ammonium acetate) because the following aminoxyTMT labeling reaction will be conducted under acidic conditions.</p><p>4. Cation exchange column was made of (30 mg) Dowex 50W-X8 resin packed on top of a 10 μL ZipTip of reversed-phase μC8 (Millipore) or 10 μL filter tip. The resins were protonated with 1 M HCl (50 μL, 3 times), washed with methanol (50 μL, 3 times), and then equilibrated with water (50 μL, 3 times) before use.</p><p>5. Apply sample solution to a cation-exchange column immediately after adding acetic acid. Otherwise, acetic acid will remove sialic acid from <i>N</i>-glycans.</p><p>6. It becomes difficult to detect the fragment ions after removing sialic acid from alditol <i>N</i>-glycan in MS/MS because of losing negative charge.</p><p>7. Removal of excess reagent was performed via solid-phase extraction using Sepharose CL4B. An organic solvent (1 mL) of 1-butanol/ethanol/water (4:1:1, v/v) was added to the dried sample containing aminoxyTMT <i>N</i>-glycans. The mixture was added to a 1.5 mL polypropylene tube containing 100 μL packed volume of Sepharose CL4B equilibrated with 1-butanol/ethanol/water (4:1:1, v/v) after activating with an aqueous solvent, ethanol/water (1:1, v/v). After gentle shaking for 30 min, the Sepharose was washed thrice with the same organic solvent (1 mL). The Sepharose was then incubated with an aqueous solvent, ethanol/water (1:1, v/v, 400 μL), for 10 min twice, and the combined liquid-phase solution (800 μL) was evaporated to dryness.</p><p>8. AminoxyTMTsixplex Label Reagent Set (aminoxyTMT6-126, 127, 128, 129, 130, and 131) have the same mass (i.e., isobaric) and chemical structure (carbonyl-reactive aminoxy group, spacer arm, and mass reporter). Nevertheless, the specific distribution of <sup>13</sup>C and <sup>15</sup>N isotopes on either side of the HCD or ETD MS/MS fragmentation site in each reagent results in a unique reporter mass (126–131 Da) in the low mass region. This set of reporter ions is used to measure the relative abundance of labeled molecules in a combined (multiplexed) MS sample representing six different treatment conditions. For glycobiology MS applications, the reagents enable quantitative profiling of glycan isoforms, such as the discovery of glycan biomarkers. However, when using HCD of LTQ Orbitrap XL, reporter ions will not be observed without aminoxyTMT <i>N</i>-glycans of ~100 pmol or more.</p></div></div><div id="g134-NglycanLCMS.References"><h2 id="_g134-NglycanLCMS_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g134-NglycanLCMS.REF.1">Nakano, M., Kakehi, K., Taniguchi, N., Kondo, A. Capillary Electrophoresis and Capillary Electrophoresis–Mass Spectrometry for Structural Analysis of <em>N</em>-Glycans Derived from Glycoproteins. In: Volpi, N. (eds) Capillary Electrophoresis of Carbohydrates. 2011. Humana Press. https://doi.org/ 10.1007/978-1-60761-875-1_9. [<a href="http://dx.crossref.org/10.1007/978-1-60761-875-1_9" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g134-NglycanLCMS.REF.2">Green ED, Adelt G, Baenziger JU, Wilson S, Van Halbeek H. The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of <em>N</em>-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy. <span><span class="ref-journal">J Biol Chem. </span>1988 Dec 5;<span class="ref-vol">263</span>(34):18253–68.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/2461366" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 2461366</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g134-NglycanLCMS.REF.3">Wilson NL, Schulz BL, Karlsson NG, Packer NH. Sequential analysis of <em>N</em>- and <em>O</em>-linked glycosylation of 2D-PAGE separated glycoproteins. <span><span class="ref-journal">J Proteome Res. </span>2002 Nov-Dec;<span class="ref-vol">1</span>(6):521–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12645620" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12645620</span></a>] [<a href="http://dx.crossref.org/10.1021/pr025538d" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g134-NglycanLCMS.REF.4">Nakano M, Saldanha R, Göbel A, Kavallaris M, Packer NH. Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells. Mol Cell Proteomics. 2011 Nov;10(11):M111.009001. doi: 10.1074/mcp.M111.009001. PMID: 21859949. [<a href="/pmc/articles/PMC3226403/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3226403</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21859949" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21859949</span></a>] [<a href="http://dx.crossref.org/10.1074/mcp.M111.009001" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g134-NglycanLCMS.REF.5">Jensen PH, Karlsson NG, Kolarich D, Packer NH. Structural analysis of <em>N</em>- and <em>O</em>-glycans released from glycoproteins. <span><span class="ref-journal">Nat Protoc. </span>2012 Jun 7;<span class="ref-vol">7</span>(7):1299–310.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/22678433" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22678433</span></a>] [<a href="http://dx.crossref.org/10.1038/nprot.2012.063" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g134-NglycanLCMS.REF.6">Kizuka Y, Kitazume S, Fujinawa R, Saito T, Iwata N, Saido TC, Nakano M, Yamaguchi Y, Hashimoto Y, Staufenbiel M, Hatsuta H, Murayama S, Manya H, Endo T, Taniguchi N. An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease. <span><span class="ref-journal">EMBO Mol Med. </span>2015 Feb;<span class="ref-vol">7</span>(2):175–89.</span> [<a href="/pmc/articles/PMC4328647/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4328647</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/25592972" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 25592972</span></a>] [<a href="http://dx.crossref.org/10.15252/emmm.201404438" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="g134-NglycanLCMS.REF.7">Kizuka Y, Nakano M, Yamaguchi Y, Nakajima K, Oka R, Sato K, Ren CT, Hsu TL, Wong CH, Taniguchi N. An Alkynyl-Fucose Halts Hepatoma Cell Migration and Invasion by Inhibiting GDP-Fucose-Synthesizing Enzyme FX, TSTA3. <span><span class="ref-journal">Cell Chem Biol. </span>2017 Dec 21;<span class="ref-vol">24</span>(12):1467–1478.e5.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/29033318" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 29033318</span></a>] [<a href="http://dx.crossref.org/10.1016/j.chembiol.2017.08.023" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="g134-NglycanLCMS.REF.8">Nakano M, Kizuka Y, Miura Y, Taniguchi N. Epigenetic regulation of neural N-glycomics. <span><span class="ref-journal">Proteomics. </span>2016 Nov;<span class="ref-vol">16</span>(22):2854–2863.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/27286656" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 27286656</span></a>] [<a href="http://dx.crossref.org/10.1002/pmic.201600053" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="g134-NglycanLCMS.REF.9">Nakano M, Mishra SK, Tokoro Y, Sato K, Nakajima K, Yamaguchi Y, Taniguchi N, Kizuka Y. Bisecting GlcNAc Is a General Suppressor of Terminal Modification of <em>N</em>-glycan. <span><span class="ref-journal">Mol Cell Proteomics. </span>2019 Oct;<span class="ref-vol">18</span>(10):2044–2057.</span> [<a href="/pmc/articles/PMC6773561/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC6773561</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/31375533" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 31375533</span></a>] [<a href="http://dx.crossref.org/10.1074/mcp.RA119.001534" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="g134-NglycanLCMS.REF.10">Pabst M, Bondili JS, Stadlmann J, Mach L, Altmann F. Mass + retention time = structure: a strategy for the analysis of <em>N</em>-glycans by carbon LC-ESI-MS and its application to fibrin <em>N</em>-glycans. <span><span class="ref-journal">Anal Chem. </span>2007 Jul 1;<span class="ref-vol">79</span>(13):5051–7.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17539604" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17539604</span></a>] [<a href="http://dx.crossref.org/10.1021/ac070363i" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="g134-NglycanLCMS.REF.11">Meitei NS, Apte A, Snovida SI, Rogers JC, Saba J. Automating mass spectrometry-based quantitative glycomics using aminoxy tandem mass tag reagents with SimGlycan. J Proteomics. 2015 Sep 8;127(Pt A):211-22. doi: 10.1016/j.jprot.2015.05.015. PMID: 26003531. [<a href="https://pubmed.ncbi.nlm.nih.gov/26003531" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 26003531</span></a>] [<a href="http://dx.crossref.org/10.1016/j.jprot.2015.05.015" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK594013_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g134-NglycanLCMS.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="g134-NglycanLCMS.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK594013/bin/g134-NglycanLCMS-Image001.jpg" alt="Figure 1: . Preparation of released N-glycans from glycoproteins and alditol reaction scheme and chemical structure of aminoxyTMT reagents and labeling reaction scheme." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Preparation of released <i>N</i>-glycans from glycoproteins and alditol reaction scheme and chemical structure of aminoxyTMT reagents and labeling reaction scheme.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g134-NglycanLCMS.F2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK594013/bin/g134-NglycanLCMS-Image002.jpg" alt="Figure 2: . Liquid chromatography-mass spectrometry (LC-MS) profiling of alditol N-glycans and aminoxyTMT N-glycans of major glycans from bovine fetuin." /></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>Liquid chromatography-mass spectrometry (LC-MS) profiling of alditol <i>N</i>-glycans and aminoxyTMT <i>N</i>-glycans of major glycans from bovine fetuin.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g134-NglycanLCMS.F3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK594013/bin/g134-NglycanLCMS-Image003.jpg" alt="Figure 3: . MS/MS of disialylated core fucosylated biantennary glycan by higher-energy C-trap dissociation (HCD) and quantification by reporter ions." /></div><h3><span class="label">Figure 3: </span></h3><div class="caption"><p>MS/MS of disialylated core fucosylated biantennary glycan by higher-energy C-trap dissociation (HCD) and quantification by reporter ions.</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: NBK594013</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/37590738" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">37590738</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/g133-MSnspectral/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/glycopodv2/g135-nucleotidesugars/" title="Next page in this title">Next ></a></div></div></div></div>
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