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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="_NBK593821_"><span class="title" itemprop="name">Preparation of glycopeptides by acetone precipitation</span></h1><div class="contrib half_rhythm"><span itemprop="author">Daisuke Takakura</span>, Dr<div class="affiliation small">Graduate School of Medical Life Science, Yokohama City University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.uc-amahokoy@arukakat_d" class="oemail">pj.ca.uc-amahokoy@arukakat_d</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">September 15, 2021</span>; Last Revision: <span itemprop="dateModified">March 14, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g7-prepacetone.Introduction"><h2 id="_g7-prepacetone_Introduction_">Introduction</h2><p>This method has been reported as an <i>N</i>-glycopeptide enrichment method from serum (<a class="bk_pop" href="#g7-prepacetone.REF.1">1</a>) (<a class="figpopup" href="/books/NBK593821/figure/g7-prepacetone.F1/?report=objectonly" target="object" rid-figpopup="figg7prepacetoneF1" rid-ob="figobg7prepacetoneF1">Figure 1</a>). It was evaluated as a rapid, simple, low-cost, and highly selective method, based on the difference in solubility of glycosylated and non-glycosylated peptides in acetone. Subsequently, it has been shown that <i>N</i>-glycopeptides can be enriched from membrane proteins (<a class="bk_pop" href="#g7-prepacetone.REF.2">2</a>) and <i>O</i>-glycopeptides from serum (<a class="bk_pop" href="#g7-prepacetone.REF.3">3</a>).</p></div><div id="g7-prepacetone.Protocol"><h2 id="_g7-prepacetone_Protocol_">Protocol</h2><p>This protocol is based on the method described in the original paper (<a class="bk_pop" href="#g7-prepacetone.REF.1">1</a>). The glycopeptide is preferentially precipitated by adding cold acetone to the digestion solution with trypsin/Lys-C, protease V8, and chymotrypsin (<a class="figpopup" href="/books/NBK593821/figure/g7-prepacetone.F2/?report=objectonly" target="object" rid-figpopup="figg7prepacetoneF2" rid-ob="figobg7prepacetoneF2">Figure 2</a>).</p><div id="g7-prepacetone.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Acetone (Cat#014-14811, FUJIFILM Wako Pure Chemical, Osaka, Japan)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Human serum, normal, 100 mL (Cat#S1-100ML, Merk, Sigma-Aldrich, St. Louis, MO, USA)</p></dd><dt>3.</dt><dd><p class="no_top_margin">Ammonium bicarbonate (Cat#012-21745, FUJIFILM Wako Pure Chemical)</p></dd><dt>4.</dt><dd><p class="no_top_margin">Pierce DTT, no-weigh format (Cat#<a href="/nuccore/2295620" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">A39255</a>, Thermo Fisher Scientific, Waltham, MA, USA)</p></dd><dt>5.</dt><dd><p class="no_top_margin">Iodoacetamide (Cat#097-05592, FUJIFILM Wako Pure Chemical)</p></dd><dt>6.</dt><dd><p class="no_top_margin">Trypsin/Lys-C mix, Mass Spec Grade (Cat#V0571, Promega, Madison, WI, USA)</p></dd></dl></div><div id="g7-prepacetone.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Centrifuge (Allegra X 30R, Beckman Coulter, Brea, CA, USA)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Temperature control and mixing (ThermoMixer C, Eppendorf, Hamburg, Germany)</p></dd></dl></div><div id="g7-prepacetone.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Preparation of protein digest</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Precipitate the protein (e.g., 50 µg) with a three-fold volume of cold acetone at −30°C overnight.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Collect the protein by centrifugation at 12,000 ×<i>g</i> at 4°C for 10 min.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Redissolve precipitated protein with 93.5 µL of 50 mM ammonium bicarbonate (pH 8.0–8.5) containing appropriate surfactant (e.g., 0.1% SDS). pH adjustment is unnecessary (<b>Note 1</b>).</p></dd><dt>d.</dt><dd><p class="no_top_margin">Add 1 µL of 0.5 M dithiothreitol (DTT) and incubate shaking with 800 × rpm at 56°C for 30 min.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Add 2.7 µL of 0.55 M iodoacetamide and incubate at room temperature in the dark for 40 min.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Add 1 µL of 0.5 M DTT and stirring immediately.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Add 1.8 µL of Trypsin/Lys-C mix solution (1.0 µg/µL) and incubate shaking with 800 × rpm at 37°C for 16 h.</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Glycopeptide enrichment with acetone (<a class="figpopup" href="/books/NBK593821/figure/g7-prepacetone.F3/?report=objectonly" target="object" rid-figpopup="figg7prepacetoneF3" rid-ob="figobg7prepacetoneF3">Figure 3</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">After digestion, place the sample tube on ice.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Add five-fold volume of cold acetone to the sample tube (<b>Note 2</b>).</p></dd><dt>c.</dt><dd><p class="no_top_margin">After mixing vigorously, incubate overnight at −30°C.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Collect the glycopeptide pellets by centrifugation at 12,000 ×<i>g</i> at 20°C for 10 min (Remove the supernatant with careful pipetting).</p></dd></dl></dd></dl></div><div id="g7-prepacetone.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Steps 1a–c may not be necessary when preparing soluble proteins, such as serum protein. Ammonium bicarbonate buffer may be added to 93.5 µL with the protein solution.</p></dd><dt>2.</dt><dd><p class="no_top_margin">An improved method to optimize the volume of cold acetone and the pH of the digestion solution has been reported using HeLa cells (<a class="bk_pop" href="#g7-prepacetone.REF.4">4</a>). Add 15 µL of 1% formic acid to 1 µL of HeLa digest (1 µg/µL). Then, HeLa glycopeptides are enriched by adding 150 µL of cold acetone.</p></dd></dl></div></div><div id="g7-prepacetone.References"><h2 id="_g7-prepacetone_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g7-prepacetone.REF.1">Takakura D, Harazono A, Hashii N, Kawasaki N. Selective glycopeptide profiling by acetone enrichment and LC/MS. <span><span class="ref-journal">J Proteomics. </span>2014 Apr 14;<span class="ref-vol">101</span>:17–30.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/24530628" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24530628</span></a>] [<a href="http://dx.crossref.org/10.1016/j.jprot.2014.02.005" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g7-prepacetone.REF.2">Takakura D, Tada M, Kawasaki N. Membrane glycoproteomics of fetal lung fibroblasts using LC/MS. <span><span class="ref-journal">Proteomics. </span>2016 Jan;<span class="ref-vol">16</span>(1):47–59.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/26439794" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 26439794</span></a>] [<a href="http://dx.crossref.org/10.1002/pmic.201500003" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g7-prepacetone.REF.3">Mancera-Arteu M, Giménez E, Benavente F, Barbosa J, Sanz-Nebot V. Analysis of <em>O</em>-glycopeptides by acetone enrichment and capillary electrophoresis-mass spectrometry. <span><span class="ref-journal">J Proteome Res. </span>2017 Nov 3;<span class="ref-vol">16</span>(11):4166–4176.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/28944674" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 28944674</span></a>] [<a href="http://dx.crossref.org/10.1021/acs.jproteome.7b00524" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g7-prepacetone.REF.4">Turiák L, Sugár S, Ács A, Tóth G, Gömöry Á, Telekes A, Vékey K, Drahos L. Site-specific <em>N</em>-glycosylation of HeLa cell glycoproteins. <span><span class="ref-journal">Sci Rep. </span>2019 Oct 15;<span class="ref-vol">9</span>(1):14822.</span> [<a href="/pmc/articles/PMC6794373/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC6794373</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/31616032" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 31616032</span></a>] [<a href="http://dx.crossref.org/10.1038/s41598-019-51428-x" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593821_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g7-prepacetone.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="g7-prepacetone.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593821/bin/g7-prepacetone-Image001.jpg" alt="Figure 1: . Strategy for serum glycoproteomics using the acetone enrichment method." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Strategy for serum glycoproteomics using the acetone enrichment method.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g7-prepacetone.F2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202%3A%20.%20Schematic%20representation%20%0200Bof%20the%20acetone%20enrichment%20method.&p=BOOKS&id=593821_g7-prepacetone-Image002.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK593821/bin/g7-prepacetone-Image002.jpg" alt="Figure 2: . Schematic representation ​of the acetone enrichment method." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>Schematic representation ​of the acetone enrichment method.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g7-prepacetone.F3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203%3A%20.%20Comparison%20of%20the%20TIC%2FXIC%20obtained%20by%20liquid%20chromatography%20with%20tandem%20mass%20spectrometry%20(LC-MS%2FMS)%20of%20the%20trypsin-digested%20top%202%20(albumin%20and%20IgG)%20removed%20human%20serum%20(1%20%B5g).&p=BOOKS&id=593821_g7-prepacetone-Image003.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK593821/bin/g7-prepacetone-Image003.jpg" alt="Figure 3: . Comparison of the TIC/XIC obtained by liquid chromatography with tandem mass spectrometry (LC-MS/MS) of the trypsin-digested top 2 (albumin and IgG) removed human serum (1 µg)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3: </span></h3><div class="caption"><p>Comparison of the TIC/XIC obtained by liquid chromatography with tandem mass spectrometry (LC-MS/MS) of the trypsin-digested top 2 (albumin and IgG) removed human serum (1 µg). (A) Control without enrichment. (B) Glycopeptide enrichment method using acetone. Because of database search using Byonic, 782 <i>N</i>-glycoforms were identified in the enriched sample (B), while 210 <i>N</i>-glycoforms were in the control (A). TIC: Total ion current, XIC: Extracted ion chromatogram.</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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