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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="_NBK593973_"><span class="title" itemprop="name">Purification method for serum free glycans</span></h1><div class="contrib half_rhythm"><span itemprop="author">Chengcheng Huang</span>, Ph.D<div class="affiliation small">Glycometabolic Biochemistry Laboratory, RIKEN CPR<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.nekir@gcgcgh" class="oemail">pj.nekir@gcgcgh</a></div></div><div class="small">Corresponding author.</div></div><div class="contrib half_rhythm"><span itemprop="author">Tadashi Suzuki</span>, Ph.D<div class="affiliation small">Glycometabolic Biochemistry Laboratory, RIKEN CPR<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.nekir@mg_ikuzust" class="oemail">pj.nekir@mg_ikuzust</a></div></div></div><p class="small">Created: <span itemprop="datePublished">November 2, 2021</span>; Last Revision: <span itemprop="dateModified">March 24, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g19-methodglycans.Introduction"><h2 id="_g19-methodglycans_Introduction_">Introduction</h2><p>Recent studies have indicated the occurrence of sialyl free <i>N</i>-glycans (FNGs) in sera (<a class="bk_pop" href="#g19-methodglycans.REF.1">1</a>,<a class="bk_pop" href="#g19-methodglycans.REF.2">2</a>). These glycans have been purified from the serum using a 3K-amicon followed by a PD-10 column and 2-aminopyridine (PA)-labeling for detecting glycans in the previous study. We optimized the glycan purification method, and using this method, we could identify glycans that were missed when using the previous amicon-purification method. Here we will introduce our optimized glycan purification method (<a class="bk_pop" href="#g19-methodglycans.REF.3">3</a>).</p></div><div id="g19-methodglycans.Protocol"><h2 id="_g19-methodglycans_Protocol_">Protocol</h2><p>This protocol first eliminates the serum proteins using ethanol precipitation, and the remaining water-soluble fractions are purified by a graphite carbon (GC) column followed by a PD-10 column, and the purified glycans are then labeled with PA for high-performance liquid chromatography (HPLC) analysis. Here the GC column is used instead of a 3K-amicon before the PD-10 purification in the previous method that was used for serum analysis (<a class="bk_pop" href="#g19-methodglycans.REF.2">2</a>).</p><div id="g19-methodglycans.Materials"><h3>Materials</h3><p>1. Ethanol (HPLC level)</p><p>2. InsertSepGC column (150 mg/3 mL, GL science, Tokyo, Japan)</p><p>3. PD-10 column (GE Healthcare, Chicago, IL)</p><p>4. 2-Aminopyridine (PA) (3 g/mL in acetic acid) (Note 1)</p><p>5. Di-methylamine-borane (0.5 mg/mL in acetic acid) (Note 2)</p><p>6. MonoFas column (GL science)</p><p>7. Elution solution: 50% acetonitrile, with 20 mM of triethylamine-acetic acid (AcOH) pH 6.0 (Note 3).</p><p>8. 5% Ethanol</p><p>9. Acetonitrile (ACN), 100% and 95%.</p><p>10. TSKgel diethylaminoethanol (DEAE)-5PW column (7.5 ϕ × 75 mm; Tosoh, Tokyo, Japan)</p><p>11. Shodex NH2P-40-3E column (3.0 mm ϕ × 250 mm, Shodex, Tokyo, Japan)</p><p>12. Inertsil ODS-3 column (2.1 ϕ × 150 mm; GL Sciences)</p><p>13. DEAE anion exchange A buffer: 10% acetonitrile containing 0.01% triethylamine.</p><p>14. DEAE anion exchange B buffer: 10% acetonitrile, with 7.4% triethylamine and 3% acetic acid.</p><p>15. Size fractionation A buffer: 93% acetonitrile in 0.3% acetate buffer (pH 7.0 adjusted by ammonia).</p><p>16. Size fractionation B buffer: 20% acetonitrile in 0.3% acetate buffer (pH 7.0 adjusted by ammonia).</p><p>17. Dual-gradient reversed-phase A buffer: 0.1 M ammonium acetate buffer, pH 6.4.</p><p>18. Dual-gradient reversed-phase B buffer: 0.1 M ammonium acetate buffer, pH 4.0 with 0.5% 1-butanol.</p><p>19. Jack bean mannosidase (JB man’ase) (Seikagaku Biobusiness Corporation, Japan).</p><p>20. Sialidase (Arthrobacter ureafaciens) (Roche Applied Science, Penzberg, Germany)</p><p>21. 10× Sialidase/JB man’ase reaction buffer: 500 mM of sodium citrate buffer, pH 4.5.</p></div><div id="g19-methodglycans.Instruments"><h3>Instruments</h3><p>1. GL science HPLC system (PUC double pumps/CO630 column oven) with a fluorescence detector (LaChrome, HITACHI High technologies, Tokyo, Japan) or equivalent.</p><p>2. pH meter</p><p>3. Vacuum evaporator</p><p>4. High-speed refrigerated centrifuge</p></div><div id="g19-methodglycans.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Pretreatment of serum</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Add 1.5 volume of ethanol to the serum, mix, and incubate on ice for 10 min (<b>Note 4</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Centrifuge at 17,000 ×<i>g</i> for 10 min at 4°C.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Take the supernatant and transfer to the new tube; dry the supernatant using a vacuum evaporator.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Dissolve the sample in 0.5–1 mL of dH<sub>2</sub>O.</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Sample desalting by GC column</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Prepare the GC column pretreated with 2 mL of 100% ACN.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Wash the column twice with 5 mL of dH<sub>2</sub>O.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Load sample on the GC column and discard the flowthrough.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Wash column twice with 5 mL of dH<sub>2</sub>O.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Elute the sample with 2.5 mL of elution solution, and collect the sample in a 15-mL tube.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Dry the eluate using a vacuum evaporator (<b>Note 5</b>).</p></dd><dt>g.</dt><dd><p class="no_top_margin">Dissolve the sample with 0.5 mL of dH<sub>2</sub>O for a PD-10 desalting.</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">A second-step desalting by PD-10</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Wash the PD-10 desalting column with 25 mL of 5% ethanol.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Load 0.5 mL of sample from Step 2g on the column.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Load 2 mL of 5% ethanol; discard the eluate.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Add 3.5 mL of 5% ethanol and collect the eluate in a new 15-mL Falcon tube.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Dry the eluate using a vacuum evaporator.</p></dd></dl></dd><dt>4.</dt><dd><p class="no_top_margin">PA-labeling</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Using the dried sample from Step 3e, redissolve the sample in 30 μL of dH<sub>2</sub>O, and dry up the sample by a vacuum evaporator (<b>Note 6</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Dissolve the sample with 20 μL of PA in AcOH. Incubate the sample at 80°C for 90 min.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add 20 μL of di-methylamine-borane in AcOH to the sample. Incubate at 80°C for another 60 min.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Wash a clean MonoFas spin column with 200 μL of dH<sub>2</sub>O, followed by 800 μL 100% ACN, and discard the eluate.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Add 460 μL of acetonitrile to the PA-labeled sample, mix, and load on the pretreated MonoFas spin column.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Centrifuge for 10,000 ×<i>g</i> for 1 min.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Wash the MonoFas spin column with 500 μL of 95% ACN, apply the solution on the MonoFas spin column, centrifuge for 10,000 ×<i>g</i> for 1 min, and discard the flowthrough.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Wash the MonoFas spin column with 800 μL of 95% ACN and discard the flowthrough.</p></dd><dt>i.</dt><dd><p class="no_top_margin">Set the spin column to a clean 1.5-mL collection tube, add 200 μL of dH<sub>2</sub>O on the column, and collect the eluate after centrifugation at 10,000 ×<i>g</i> for 1 min.</p></dd></dl></dd><dt>5.</dt><dd><p class="no_top_margin">Detection of glycans by HPLC (<a class="bk_pop" href="#g19-methodglycans.REF.4">4</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Treat sample with sialidase to identify sialylated free glycans.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Apply the sample to DEAE anion exchange HPLC (DEAE-5PW column) (<a class="bk_pop" href="#g19-methodglycans.REF.3">3</a>).</p></dd><dt>c.</dt><dd><p class="no_top_margin">HPLC elution conditions and detection of the PA-labeled glycans are as follows:</p><p>Gradient program (% of DEAE anion exchange B buffer, 1 mL/min, at 25°C):</p><p>0–5 min, isocratic 0%;</p><p>5–45 min, 0%–20%;</p><p>45–50 min, isocratic 100%;</p><p>50–60 min, isocratic 0%.</p><p>Emission wavelength: 380 nm and Excitation wavelength: 310 nm.</p></dd><dt>d.</dt><dd><p class="no_top_margin">For sialyl free glycans, collect sialidase-sensitive peaks for further analysis (<a class="figpopup" href="/books/NBK593973/figure/g19-methodglycans.F1/?report=objectonly" target="object" rid-figpopup="figg19methodglycansF1" rid-ob="figobg19methodglycansF1">Figure 1</a>) (<b>Note 7</b>).</p></dd><dt>e.</dt><dd><p class="no_top_margin">For the analysis of neutral glycans, collect the flowthrough fraction of the DEAE HPLC and apply to a size fractionation HPLC (NH2P-40-3E column) with the following elution conditions (% of size fractionation B buffer, 0.45 mL/min at 25°C):</p><p>0–0.5 min, 1%–10%;</p><p>0.5–3 min, 10%–25%;</p><p>3–33 min, 25%–55%;</p><p>33.1–35 min, isocratic 70%;</p><p>35.1–55 min, isocratic 1%.</p><p>Emission wavelength: 380 nm and Excitation wavelength: 310 nm.</p></dd><dt>f.</dt><dd><p class="no_top_margin">The peaks can be further separated by dual-gradient, reversed-phase HPLC (<a class="bk_pop" href="#g19-methodglycans.REF.5">5</a>) for structural characterization (<a class="figpopup" href="/books/NBK593973/figure/g19-methodglycans.F2/?report=objectonly" target="object" rid-figpopup="figg19methodglycansF2" rid-ob="figobg19methodglycansF2">Figure 2</a>) (<a class="bk_pop" href="#g19-methodglycans.REF.3">3</a>) (<b>Note 8</b>).</p><p>The elution condition (% of dual-gradient reversed-phase B buffer, 0.2 mL/min at 25°C):</p><p>0–10 min, isocratic 1%;</p><p>10–110 min, 1%–70%;</p><p>110.1–120 min, isocratic 70%;</p><p>120.1–150 min, isocratic 1%.</p><p>Emission wavelength: 400 nm and Excitation wavelength: 320 nm.</p></dd></dl></dd></dl></div><div id="g19-methodglycans.Notes"><h3>Notes</h3><p>1. Make PA freshly or store at −30°C and use within a month.</p><p>2. Make freshly.</p><p>3. Forty percent ACN is sufficient to elute FNGs smaller than tri-sialyl FNGs, while it is essential to increase the ACN to 50% to elute tetra-sialyl FNGs. The increase in ACN will also elute unwanted salt, which influences the result, so it is recommended to maintain the ACN at a low concentration if the sample is expected to contain FNGs smaller than tri-sialyl glycans.</p><p>4. Serum with volume from 100 μL to 1 mL could be used for this analysis. The incubation time is critical for analysis as longer incubation will cause precipitation of the glycans.</p><p>5. Add 200 μL of dH2O, and dry the sample once again, if there is still some salt remaining in the tube.</p><p>6. Dry the sample completely. The remaining water may cause enhanced epimerization upon PA-labeling, which could artificially change the reducing-terminal glycan during the labeling process.</p><p>7. The condition for sialidase treatment is as follows: Add 10 μL of the PA-labeled sample with 1 μL of the reaction buffer and 0.5 μL of Sialidase and incubate overnight at 37°C. After the reaction, boil the reaction solution at 95°C for 5 min, precipitate the protein as Steps 1a–c, and redissolve it in 10 μL of dH<sub>2</sub>O for HPLC analysis.</p><p>8. The condition for JB man’ase treatment is the same as the sialidase treatment, except the fact that JB man’ase is added instead of sialidase.</p></div></div><div id="g19-methodglycans.References"><h2 id="_g19-methodglycans_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g19-methodglycans.REF.1">Iwatsuka K, Watanabe S, Kinoshita M, Kamisue K, Yamada K, Hayakawa T, Suzuki T, Kakehi K. Free glycans derived from glycoproteins present in human sera. <span><span class="ref-journal">J Chromatogr B Analyt Technol Biomed Life Sci. </span>2013 Jun 1;<span class="ref-vol">928</span>:16–21.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/23584042" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23584042</span></a>] [<a href="http://dx.crossref.org/10.1016/j.jchromb.2013.03.010" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g19-methodglycans.REF.2">Seino J, Fujihira H, Nakakita SI, Masahara-Negishi Y, Miyoshi E, Hirabayashi J, Suzuki T. Occurrence of free sialyl oligosaccharides related to N-glycans (sialyl free N-glycans) in animal sera. <span><span class="ref-journal">Glycobiology. </span>2016 Oct;<span class="ref-vol">26</span>(10):1072–1085.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/27102284" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 27102284</span></a>] [<a href="http://dx.crossref.org/10.1093/glycob/cww048" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g19-methodglycans.REF.3">Huang C, Seino J, Fujihira H, Sato K, Fujinawa R, Sumer-Bayraktar Z, Ishii N, Matsuo I, Nakaya S, Suzuki T. Occurrence of free N-glycans with a single GlcNAc at the reducing termini in animal sera. Glycobiology. 2021 Dec 3:cwab124. doi: 10.1093/glycob/cwab124. PMID: 34939097. [<a href="https://pubmed.ncbi.nlm.nih.gov/34939097" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 34939097</span></a>] [<a href="http://dx.crossref.org/10.1093/glycob/cwab124" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g19-methodglycans.REF.4">Hirayama H, Seino J, Kitajima T, Jigami Y, Suzuki T. Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae. <span><span class="ref-journal">J Biol Chem. </span>2010 Apr 16;<span class="ref-vol">285</span>(16):12390–404.</span> [<a href="/pmc/articles/PMC2852977/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2852977</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20150426" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20150426</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M109.082081" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g19-methodglycans.REF.5">Suzuki T, Matsuo I, Totani K, Funayama S, Seino J, Taniguchi N, Ito Y, Hase S. Dual-gradient high-performance liquid chromatography for identification of cytosolic high-mannose-type free glycans. <span><span class="ref-journal">Anal Biochem. </span>2008 Oct 15;<span class="ref-vol">381</span>(2):224–32.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18656438" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18656438</span></a>] [<a href="http://dx.crossref.org/10.1016/j.ab.2008.07.002" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593973_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g19-methodglycans.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="g19-methodglycans.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593973/bin/g19-methodglycans-Image001.jpg" alt="Figure 1: . Diethylaminoethanol (DEAE) anion-exchange high-performance liquid chromatography HPLC of the GC purified adult bovine serum compared with the previous purification method by amicon (2)." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Diethylaminoethanol (DEAE) anion-exchange high-performance liquid chromatography HPLC of the GC purified adult bovine serum compared with the previous purification method by amicon (<a class="bk_pop" href="#g19-methodglycans.REF.2">2</a>, <a class="bk_pop" href="#g19-methodglycans.REF.3">3</a>). Upper two panels, GC column purified adult bovine serum and the sialidase-treated control. Lower two panels, the previous Amicon (<a class="bk_pop" href="#g19-methodglycans.REF.2">2</a>) purification method purified sample and sialidase-treated control. Sialidase treated sample was analyzed simultaneously to show the sialyl glycans; note the small-sia glycans in the GC column purified sample that could not be detected by the amicon method.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g19-methodglycans.F2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593973/bin/g19-methodglycans-Image002.jpg" alt="Figure 2: . Dual-gradient reversed-phase high-performance liquid chromatography (HPLC) of flowthrough (FL) fraction from the diethylaminoethanol (DEAE) anion-exchange HPLC from GC column purified adult bovine serum (upper) and JB man’ase-treated control (lower) (3)." /></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>Dual-gradient reversed-phase high-performance liquid chromatography (HPLC) of flowthrough (FL) fraction from the diethylaminoethanol (DEAE) anion-exchange HPLC from GC column purified adult bovine serum (upper) and JB man’ase-treated control (lower) (<a class="bk_pop" href="#g19-methodglycans.REF.3">3</a>). The oligomannose-type FNGs peaks sensitive to JB man’ase treatment are indicated by black arrows, and the elution position of ManGlcNAc and ManGlcNAc<sub>2</sub> after the JB Man’ase are shown by dotted lines. Green circle, mannose (Man); blue square, <i>N</i>-acetylglucosamine (GlcNAc).</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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