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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="_NBK593967_"><span class="title" itemprop="name">Structural characterization of glycosphingolipids by liquid chromatography-mass spectrometry</span></h1><div class="contrib half_rhythm"><span itemprop="author">Akemi Suzuki</span>, Dr<div class="affiliation small">Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.en.nco.aeag@kzsmka" class="oemail">pj.en.nco.aeag@kzsmka</a></div></div><div class="small">Corresponding author.</div></div><div class="contrib half_rhythm"><span itemprop="author">Takahiro Nitta</span>, Dr.<div class="affiliation small">Institute of Molecular Biomembrane and Glycobiology,
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Tohoku Medical and Pharmaceutical University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.upm-ukohot@attin.t" class="oemail">pj.ca.upm-ukohot@attin.t</a></div></div></div><div class="contrib half_rhythm"><span itemprop="author">Atit Silsirivanit</span>, Dr.<div class="affiliation small">Faculty of Medicine. Khon Kaen University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ht.ca.ukk@listita" class="oemail">ht.ca.ukk@listita</a></div></div></div><div class="contrib half_rhythm"><span itemprop="author">Naoto Oikawa</span>, Dr.<div class="affiliation small">Institute of Genetic Medicine, Hokkaido University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.iadukoh.mgi@awakiOotoaN" class="oemail">pj.ca.iadukoh.mgi@awakiOotoaN</a></div></div></div><div class="contrib half_rhythm"><span itemprop="author">Junko Matsuda</span>, Dr.<div class="affiliation small">Department of Pathophysiology and Metabolism, Kawasaki Medica School<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.m-ikasawak.dem@nujustam" class="oemail">pj.ca.m-ikasawak.dem@nujustam</a></div></div></div><div class="contrib half_rhythm"><span itemprop="author">Jin-ichi Inokuchi</span>, Dr.<div class="affiliation small">Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.upm-ukohot@nij" class="oemail">pj.ca.upm-ukohot@nij</a></div></div></div><p class="small">Created: <span itemprop="datePublished">September 25, 2021</span>; Last Revision: <span itemprop="dateModified">February 27, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g141-glycosphinLC-MS.Introduction"><h2 id="_g141-glycosphinLC-MS_Introduction_">Introduction</h2><p>Nuclear magnetic resonance and mass spectrometry (MS) are fundamental analytical methods for the structural characterization of glycosphingolipids (GSLs). Liquid chromatography–mass spectrometry (LC-MS) is a method that can be applied to crude GSL fractions prepared from biological materials without further extensive purification. Thin-layer chromatography, with chemical detection and binding assays using antibodies and carbohydrate recognition reagents, is an excellent and low-cost procedure. It has become indispensable because it can provide both an overview of GSL composition and information about the carbohydrate structure with consuming small amounts of samples. However, the functions of GSLs in membrane microdomains have attracted attention (<a class="bk_pop" href="#g141-glycosphinLC-MS.REF.1">1</a>, <a class="bk_pop" href="#g141-glycosphinLC-MS.REF.2">2</a>). For the microdomain research, the structural information on the lipid part of GSLs, ceramides, is critical. MS can provide structural information on ceramides and the glycan structures of GSLs in crude lipid preparations. This protocol describes liquid chromatography–electrospray ionization/MS (LC–ESI/MS) for the structural characterization of GSLs (<a class="bk_pop" href="#g141-glycosphinLC-MS.REF.3">3</a>–<a class="bk_pop" href="#g141-glycosphinLC-MS.REF.11">11</a>). Despite recent significant developments in LC-MS technology, several challenges still remain in the use of this method for GSL analysis, which are discussed in the Notes section.</p></div><div id="g141-glycosphinLC-MS.Protocol"><h2 id="_g141-glycosphinLC-MS_Protocol_">Protocol</h2><p>This protocol focuses on the structural characterization of GSLs using LC–ESI/MS with reversed-phase chromatography and hydrophilic interaction liquid chromatography (HILIC).</p><div id="g141-glycosphinLC-MS.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Reagents: water, methanol, isopropanol, acetonitrile, formic acid, ammonium formate, acetic acid (all LC-MS or MS grade), ammonia water (25 or 28% reagent grade), and stock solutions of 0.4 M and 0.1 M ammonium formate (stored at 4°C for at most 6 months).</p></dd><dt>2.</dt><dd><p class="no_top_margin">Columns: A C30 reversed-phase column (Develosil C30, 1 × 50 mm, Nomura Chemical, Japan, or equivalent) for reversed-phase LC–ESI/MS, an NH<sub>2</sub> HILIC column (Inertsil NH<sub>2</sub> column 1 mm i.d. × 50 mm, GL Science, Japan) for normal-phase LC–ESI/MS.</p></dd><dt>3.</dt><dd><p class="no_top_margin">Solvents for reversed-phase LC-MS: Solvent A: 0.1 mL of acetic acid/0.1 mL of ammonia water/25 mL of water/25 mL of methanol/50 mL of isopropanol. Solvent B: 0.1 mL of acetic acid/0.1 mL of ammonia water/2 mL of water/48 mL of methanol/50 mL of isopropanol.</p></dd><dt>4.</dt><dd><p class="no_top_margin">Solvents for normal-phase HILIC LC-MS: Solvent C: 0.1 mL of 0.1 M ammonium formate/83 mL of acetonitrile/16.9 mL of water. Solvent D: 50 mL of 0.1 M ammonium formate/50 mL of acetonitrile.</p></dd><dt>5.</dt><dd><p class="no_top_margin">Samples of crude GSL mixtures: Even though samples subjected to LC-MS analysis can be crude GSL mixtures prepared from cells and tissues, the GSL content and contamination by phospholipids and other molecules are critical issues in terms of obtaining sufficient information for the structural characterization. Mild alkaline hydrolysis and the application of C18 cartridges to remove salts and polar contaminants are recommended. The protocols used for preparing crude GSL mixtures are described in this GlycoPOD.</p></dd></dl></div><div id="g141-glycosphinLC-MS.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">A mass spectrometer with an ESI ion source and an ion trap for achieving MS<sup>n</sup> or MS<sup>2</sup>. For MS<sup>2</sup>, three scans are acquired from m/z 200 to 2,000 with an accumulation time of 50 ms. For MS<sup>3</sup>, three scans are acquired from m/z 100 to 700. An MS instrument capable of MS<sup>n</sup> or MS<sup>2</sup> analysis should be set up in accordance with the manufacturer’s protocol. A mass spectrometer with a measurement accuracy of less than 50 ppm is recommended.</p></dd><dt>2.</dt><dd><p class="no_top_margin">An LC with an autosampler, which is capable of a flow rate of 0.05 mL/min and programed elution.</p></dd><dt>3.</dt><dd><p class="no_top_margin">The following MS parameters are used by the authors for a routine analysis and should be changed according to the instructions of individual MS instruments. Three independent scans of each mass spectrum from m/z 500 to 2,000 were conducted with an accumulation time of 50 ms. Spectra were acquired in the negative-ion mode with a detector voltage of 1.9 kV or equivalent. The collision-induced dissociation energy for MS<sup>2</sup> and MS<sup>3</sup> and the collision gas flow of Ar should be held at their 50% arbitrary values or optimized using standard GSLs. The temperature of the curved desolvation device should be 200°C. These parameters can be modified according to the protocols of individual MS instruments after being optimized by several trials or following a successful routine analysis.</p></dd><dt>4.</dt><dd><p class="no_top_margin">A sonic bath; any model is suitable.</p></dd></dl></div><div id="g141-glycosphinLC-MS.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">LC-MS with separation using a C30 reversed-phase column (<a class="bk_pop" href="#g141-glycosphinLC-MS.REF.5">5</a>, <a class="bk_pop" href="#g141-glycosphinLC-MS.REF.9">9</a>, <a class="bk_pop" href="#g141-glycosphinLC-MS.REF.10">10</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">The LC is set up as follows: a C-30 column as described above; flow rate of 0.05 mL/min; column oven temperature of 25°C; solvents A and B; programed elution is 0% solvent B in solvent A for 5 min, 0%–100% solvent B for 30 min, 100% solvent B for 10 min, and 0% solvent B for 10 min; and isopropanol is used as a washing solvent for the autosampler (<b>Note 1</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">The MS is set up as follows: MS<sup>2</sup> scanning in the negative-ion mode by an autoprogram provided by the MS instrument manufacturer, and collision energy (CE) is the default value or is defined after several runs to determine the optimal conditions. If the m/z values of the GSLs of interest are already known or are provided by the autoprogram, the MS<sup>2</sup> analysis with particular m/z values as the precursor ions is conducted to obtain better quality MS<sup>2</sup> spectra. If an MS<sup>3</sup> analysis is possible, the m/z values of ceramide fragment ions are selected as the precursor ions (<b>Note 2</b>).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Samples of crude GSL fractions are dissolved in the minimum volume of methanol or solvent A. The injection volume should be less than 10 μL. Sonication in a sonic bath is used to agitate sample solutions. The temperature of the autosampler is set at 4°C. Run an analysis (<b>Note 3</b>).</p></dd><dt>d.</dt><dd><p class="no_top_margin">Data mining of structural analysis. <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F1/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF1" rid-ob="figobg141glycosphinLCMSF1">Figure 1</a> shows the annotation of fragment ions of GM3 as an example and a m/z list of the fragments derived from the ceramides in the negative-ion mode analysis.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Data mining of glycan structures. <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F2/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF2" rid-ob="figobg141glycosphinLCMSF2">Figure 2A</a> shows selected mass chromatograms of seven major molecular species of Gb4Cer as an example of a neutral GSL analysis. Glycan sequences of GSLs are confirmed by MS<sup>2</sup>, and the neutral losses are used to identify Hex as 162, HexNAc as 203, and NeuNAc as 291. <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F2/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF2" rid-ob="figobg141glycosphinLCMSF2">Figure 2B</a> shows the MS<sup>2</sup> spectrum obtained at the * marked peak with m/z 1337.87 as the precursor ion, and the MS<sup>2</sup> spectrum identifies the molecule to be Gb4Cer by detecting 648 Y<sub>0</sub>, 810 Y<sub>1</sub>, 972 Y<sub>2</sub>, and 1134 Y<sub>3</sub> fragments and supporting the calculation of 648(162)810(162)972(162 + 203)1337. With the results of data mining of the ceramide structure described in step 1g of Methods, the molecule is finally identified to be Gb4Cer(d18:1-24:0). The structures of other Gb4Cer molecular species should be identified in the same way as Gb4Cer(d18:1-24:0) (<b>Note 4</b>).</p></dd><dt>f.</dt><dd><p class="no_top_margin">Data mining of glycan structures. <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F2/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF2" rid-ob="figobg141glycosphinLCMSF2">Figure 2D</a> shows the mass chromatograms of seven major molecular species of GM3 as an example of an acidic GSL or ganglioside analysis. <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F2/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF2" rid-ob="figobg141glycosphinLCMSF2">Figure 2E</a> shows the MS<sup>2</sup> spectrum obtained at the * marked peak with m/z 1263.83 as the precursor ion. The MS<sup>2</sup> spectrum identifies the molecule to be GM3 by detecting 648 Y<sub>0</sub>, 810 Y<sub>1</sub>, and 972 Y<sub>2</sub> fragments and supporting the calculation of 648(162)810(162)972(291)1263. With the results of the data mining of ceramide structures, the molecule is identified to be GM3(d18:1-24:0). The structures of other GM3 molecular species should be identified in the same manner. Mass chromatograms of Gb4Cer and GM3 shown in <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F2/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF2" rid-ob="figobg141glycosphinLCMSF2">Figures 2A</a> and <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F2/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF2" rid-ob="figobg141glycosphinLCMSF2">2D</a> were obtained using the reversed-phase LC separation and, therefore, the retention times of the peaks should also be taken into consideration for confirming the correct structural identification.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Data mining of ceramide structures. <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F2/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF2" rid-ob="figobg141glycosphinLCMSF2">Figures 2C</a> and <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F2/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF2" rid-ob="figobg141glycosphinLCMSF2">2F</a> show the MS<sup>2</sup> spectra of Gb4Cer and of GM3 covering the low m/z region from m/z 200 to 500. Fragments P and R are derived from sphingosine and V to S are derived from acyl chains carrying parts of sphingosine. These fragment ions are used to identify ceramide structures (<b>Note 5</b>).</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">LC-MS with the separation by a NH<sub>2</sub> HILIC column (<a class="bk_pop" href="#g141-glycosphinLC-MS.REF.4">4</a>, <a class="bk_pop" href="#g141-glycosphinLC-MS.REF.7">7</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">The LC is set up as follows: an NH<sub>2</sub> HILIC column as described above; flow rate of 0.05 μL/min; column oven temperature of 25°C; solvent C and D; and the programed elution is 0% solvent D in solvent C for 5 min, from 0% to 76% solvent D in solvent C for 15 min, from 76% to 90% solvent D in solvent C for 5 min, and 90% solvent D in solvent C for 10 min (<a class="bk_pop" href="#g141-glycosphinLC-MS.REF.4">4</a>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">The MS is set up as follows: MS<sup>2</sup> scanning in the negative-ion mode using an autoprogram provided by the MS instrument manufacturer. The CE is the default value or is defined after several runs to determine the optimal conditions. An MS<sup>2</sup> analysis with particular m/z values is conducted to obtain the better quality MS<sup>2</sup> spectra. If an MS<sup>3</sup> analysis is possible, the m/z values of ceramide fragment ions are selected as the precursor ions.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Dissolve samples of crude GSL fractions in the minimum volume of solvent C. The injection volume should be less than 10 μL. Sonication in a sonic bath is used to agitate sample solutions. The temperature of the autosampler is set to 4°C. Run an analysis.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Data mining of the structural analysis. Refer to <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F1/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF1" rid-ob="figobg141glycosphinLCMSF1">Figure 1</a> for the annotation and m/z of fragment ions. <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F3/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF3" rid-ob="figobg141glycosphinLCMSF3">Figure 3</a> shows a ganglioside analysis of synaptosomes prepared from the human brain (<a class="bk_pop" href="#g141-glycosphinLC-MS.REF.7">7</a>), focusing on the separation of GD1a and GD1b molecules.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Data mining of glycan structure. <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F3/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF3" rid-ob="figobg141glycosphinLCMSF3">Figure 3A</a> shows mass chromatograms of six major gangliosides of the synaptosomes. <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F3/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF3" rid-ob="figobg141glycosphinLCMSF3">Figures 3B</a> and <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F3/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF3" rid-ob="figobg141glycosphinLCMSF3">3C</a> show MS<sup>2</sup> spectra with m/z 931 as the precursor ion obtained at two different retention times, 16 min for peak B, and 17 min for peak C. The MS<sup>2</sup> spectra detect Y<sub>0</sub>–Y<sub>2</sub> as ceramide-carrying glycan fragment ions, m/z 290 for NeuNAc, and 581 for NeuNAc-NeuNAc. The presence of m/z 290, the absence of m/z 581 in peak B, and the presence of m/z 290 and 581 in peak C clearly demonstrate that peak B does not contain NeuNAc-NeuNAc and peak C contains NeuNAc-NeuNAc sequence, confirming that peak B is GD1a and peak C is GD1b.</p></dd></dl></dd></dl></div><div id="g141-glycosphinLC-MS.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">The size of C30 columns can be changed. Then, the flow rate and the program of gradient elution are optimized.</p></dd><dt>2.</dt><dd><p class="no_top_margin">If an MS<sup>3</sup> analysis is not available, MS<sup>2</sup> will still detect fragment ions derived from ceramide. <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F4/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF4" rid-ob="figobg141glycosphinLCMSF4">Figure 4</a> shows an example of this MS<sup>2</sup> analysis. A higher MS<sup>2</sup> CE is required for this analysis (<a class="bk_pop" href="#g141-glycosphinLC-MS.REF.10">10</a>). A negative-ion mode analysis provides more structural information than a positive-ion mode analysis.</p></dd><dt>3.</dt><dd><p class="no_top_margin">The quality of samples of GSL mixtures, even as crude mixtures, is critical because the suppression of ionization, the saturation of ion detection, and other effects will limit the quality of the mass spectra of the target GSL molecules.</p></dd><dt>4.</dt><dd><p class="no_top_margin">In this analysis, the presence of adduct ions is a critical drawback. The elution solvents contain acetic acid as an ionization-enhancing matrix and produce an adduct of [M + CH<sub>3</sub>COO]<sup>−</sup>. There is a 60 mass unit difference corresponding to 18(H<sub>2</sub>O) + 16(O) + 26(CH=CH), which can affect cases wherein discrimination is required between GSLs carrying ceramides of d18:1-normal fatty acid with n carbon number (named M1) and t18:0-hydroxy fatty acid with one double bond and n + 2 carbon number (named M2). The m/z of M1 + 60 is equal to that of M2. Careful attention is required to analyze mass chromatograms when focusing on GSLs carrying trihydroxysphinganines and hydroxy fatty acids. Another drawback is that the amount of [M-H]<sup>−</sup> ion is reduced due to this adduct formation. Unfortunately, formic acid is often used as a matrix and produces an adduct of [M + HCOO]<sup>−</sup>. Gangliosides do not produce these adducts.</p></dd><dt>5.</dt><dd><p class="no_top_margin">If an MS<sup>3</sup> analysis is available, the ceramide structures are analyzed with confidence. However, it is often the case that the signal intensity of ceramide precursor ions for MS<sup>3</sup> analysis is weak, and then MS<sup>2</sup> analysis focusing on ceramide-derived fragments is worth attempting (<a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F4/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF4" rid-ob="figobg141glycosphinLCMSF4">Figure 4</a>).</p></dd></dl></div></div><div id="g141-glycosphinLC-MS.References"><h2 id="_g141-glycosphinLC-MS_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g141-glycosphinLC-MS.REF.1">Simons K, Ikonen E. Functional rafts in cell membranes. <span><span class="ref-journal">Nature. </span>1997 Jun 5;<span class="ref-vol">387</span>(6633):569–72.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9177342" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9177342</span></a>] [<a href="http://dx.crossref.org/10.1038/42408" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g141-glycosphinLC-MS.REF.2">Aureli M, Grassi S, Sonnino S, Prinetti A. Isolation and analysis of detergent-resistant membrane fractions. <span><span class="ref-journal">Methods Mol Biol. </span>2016;<span class="ref-vol">1376</span>:107–31.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/26552679" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 26552679</span></a>] [<a href="http://dx.crossref.org/10.1007/978-1-4939-3170-5_10" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g141-glycosphinLC-MS.REF.3">Ikeda K, Shimizu T, Taguchi R. Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-MS/MS with theoretically expanded multiple reaction monitoring. <span><span class="ref-journal">J Lipid Res. </span>2008 Dec;<span class="ref-vol">49</span>(12):2678–89.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18703820" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18703820</span></a>] [<a href="http://dx.crossref.org/10.1194/jlr.D800038-JLR200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g141-glycosphinLC-MS.REF.4">Ikeda K, Taguchi R. Highly sensitive localization analysis of gangliosides and sulfatides including structural isomers in mouse cerebellum sections by combination of laser microdissection and hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry with theoretically expanded multiple reaction monitoring. <span><span class="ref-journal">Rapid Commun Mass Spectrom. </span>2010 Oct 30;<span class="ref-vol">24</span>(20):2957–65.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/20872628" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20872628</span></a>] [<a href="http://dx.crossref.org/10.1002/rcm.4716" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g141-glycosphinLC-MS.REF.5">Nagafuku M, Okuyama K, Onimaru Y, Suzuki A, Odagiri Y, Yamashita T, Iwasaki K, Fujiwara M, Takayanagi M, Ohono I, Inokuchi J. CD4 and CD8 T cells require different membrane gangliosides for activation. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2012 Feb 7;<span class="ref-vol">109</span>(6):E336–42.</span> [<a href="/pmc/articles/PMC3277553/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3277553</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22308377" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22308377</span></a>] [<a href="http://dx.crossref.org/10.1073/pnas.1114965109" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g141-glycosphinLC-MS.REF.6">Ito E, Waki H, Miseki K, Shimada T, Sato T, Kakehi K, Suzuki M, Suzuki A. Structural characterization of neutral glycosphingolipids using high-performance liquid chromatography-mass spectrometry with a repeated high-speed polarity and MS<sup>n</sup> switching system. <span><span class="ref-journal">Glycoconj J. </span>2013 Dec;<span class="ref-vol">30</span>(9):881–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/23959431" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23959431</span></a>] [<a href="http://dx.crossref.org/10.1007/s10719-013-9492-8" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="g141-glycosphinLC-MS.REF.7">Oikawa N, Hatsuta H, Murayama S, Suzuki A, Yanagisawa K. Influence of APOE genotype and the presence of Alzheimer pathology on synaptic membrane lipids of human brains. <span><span class="ref-journal">J Neurosci Res. </span>2014 May;<span class="ref-vol">92</span>(5):641–50.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/24446209" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24446209</span></a>] [<a href="http://dx.crossref.org/10.1002/jnr.23341" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="g141-glycosphinLC-MS.REF.8">Oikawa N, Matsubara T, Fukuda R, Yasumori H, Hatsuta H, Murayama S, Sato T, Suzuki A, Yanagisawa K. Imbalance in fatty-acid-chain length of gangliosides triggers Alzheimer amyloid deposition. PLoS One. 2015 Mar 23;10(3):e0121356. doi: 10.1371/journal.pone.0121356. eCollection 2015. PMID: 25798597. [<a href="/pmc/articles/PMC4370507/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4370507</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/25798597" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 25798597</span></a>] [<a href="http://dx.crossref.org/10.1371/journal.pone.0121356" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="g141-glycosphinLC-MS.REF.9">Veillon L, Go S, Matsuyama W, Suzuki A, Nagasaki M, Yatomi Y, Inokuchi J. Identification of ganglioside GM3 molecular species in human serum associated with risk factors of metabolic syndrome. <span><span class="ref-journal">PLoS One. </span>2015 Jun 23;<span class="ref-vol">10</span>(6):e0129645. </span> [<a href="/pmc/articles/PMC4477979/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4477979</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/26102277" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 26102277</span></a>] [<a href="http://dx.crossref.org/10.1371/journal.pone.0129645" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="g141-glycosphinLC-MS.REF.10">Silsirivanit A, Phoomak C, Teeravirote K, Wattanavises S, Seubwai W, Saengboonmee C, Zhan Z, Inokuchi J, Suzuki A, Wongkham S. Overexpression of HexCer and LacCer containing 2-hydroxylated fatty acids in cholangiocarcinoma and the association of the increase of LacCer (d18:1-h23:0) with shorter survival of the patients. <span><span class="ref-journal">Glycoconj J. </span>2019 Apr;<span class="ref-vol">36</span>(2):103–111.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/30888588" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 30888588</span></a>] [<a href="http://dx.crossref.org/10.1007/s10719-019-09864-4" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="g141-glycosphinLC-MS.REF.11">Tanaka K, Suzuki A, Aoki D, Iwamori M. Characterization of a novel glycolipid with a difucosylated H-antigen in human blood group O erythrocytes with monoclonal antibody HMMC-1 and its detection in human uterine cervical carcinoma tissues. <span><span class="ref-journal">Glycoconj J. </span>2019 Jun;<span class="ref-vol">36</span>(3):219–226.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/31098851" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 31098851</span></a>] [<a href="http://dx.crossref.org/10.1007/s10719-019-09873-3" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593967_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g141-glycosphinLC-MS.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="g141-glycosphinLC-MS.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593967/bin/g141-glycosphinLC-MS-Image001.jpg" alt="Figure 1: . Annotation of fragment ions used for the structural characterization of glycosphingolipids (GSLs)." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Annotation of fragment ions used for the structural characterization of glycosphingolipids (GSLs). A: The fragmentation of GM3 as an example for a GSL. B: m/z lists of reporter fragment signals containing acyl chains and sphingosines in the negative-ion mode analysis.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g141-glycosphinLC-MS.F2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593967/bin/g141-glycosphinLC-MS-Image002.jpg" alt="Figure 2: . Structural characterization of Gb4Cer(d18:1-24:0) and GM3(d18:1-24:0) using liquid chromatography–mass spectrometry with a C30 reversed-phase column." /></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>Structural characterization of Gb4Cer(d18:1-24:0) and GM3(d18:1-24:0) using liquid chromatography–mass spectrometry with a C30 reversed-phase column. A: Selected ion-mass chromatograms of Gb4Cer. B: Peak * with m/z 1337.87 was subjected to MS<sup>2</sup> analysis. C: The resulting ceramide fragment ion with m/z 648.63 was subjected to MS<sup>3</sup> analysis. Based on these results, peak * is identified to be Gb4(d18:1-24:0). D: Selected ion-mass chromatograms of GM3. E: Peak * with m/z 1263.83 was subjected to MS<sup>2</sup> analysis. F: The resulting ceramide fragment ion with m/z 648.63 was subjected to MS<sup>3</sup> analysis. Based on these results, peak * is identified to be GM3(d18:1-24:0). Annotation for the assignments of reporter signals is shown in <a class="figpopup" href="/books/NBK593967/figure/g141-glycosphinLC-MS.F1/?report=objectonly" target="object" rid-figpopup="figg141glycosphinLCMSF1" rid-ob="figobg141glycosphinLCMSF1">Figure 1</a>.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g141-glycosphinLC-MS.F3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593967/bin/g141-glycosphinLC-MS-Image003.jpg" alt="Figure 3: . Characterization of glycan chains of GD1a and 1b using liquid chromatography–mass spectrometry with a NH2-hydrophilic interaction liquid chromatography (HILIC) column." /></div><h3><span class="label">Figure 3: </span></h3><div class="caption"><p>Characterization of glycan chains of GD1a and 1b using liquid chromatography–mass spectrometry with a NH<sub>2</sub>-hydrophilic interaction liquid chromatography (HILIC) column. A: Selected mass chromatograms of major gangliosides of human synaptosomes, focusing on the separation of GD1a and GD1b. B: The MS<sup>2</sup> spectrum of peak B detected m/z 290.10 derived from NeuNAc but did not detect m/z 581.18 derived from NeuNAc-NeuNAc, indicating the presence of NeuNAc- linkage but not a NeuNAc-NeuNAc linkage. C: The MS<sup>2</sup> spectrum of peak C detected m/z 290.10 and 581.18, indicating the presence of NeuNAc-NeuNAc linkage. Y<sub>1</sub>–Y<sub>5</sub> are glycan carrying fragments, and Y<sub>0</sub> is a ceramide fragment.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g141-glycosphinLC-MS.F4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593967/bin/g141-glycosphinLC-MS-Image004.jpg" alt="Figure 4: . Structural characterization of LacCer using an MS2 analysis." /></div><h3><span class="label">Figure 4: </span></h3><div class="caption"><p>Structural characterization of LacCer using an MS<sup>2</sup> analysis. If an MS<sup>3</sup> analysis is unavailable, an MS<sup>2</sup> analysis can detect some but not all of the fragments derived from ceramide and is still able to define ceramide structures. The upper panel shows selected mass chromatograms of LacCers in human sera, and the lower panels of MS<sup>2</sup> spectra detect Hex-Cer (Y<sub>1</sub>), Cer (Y<sub>0</sub>), and ceramide-derived fragments, which characterize the structures of LacCer molecular species.</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: NBK593967</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/37590699" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">37590699</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/g140-Ionmobilitymass/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/glycopodv2/g142-conglycoliplcms/" title="Next page in this title">Next ></a></div></div></div></div>
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