<html> 1。酶法deglycosylation <ol><li>使用PCR管中，以尽量减少因蒸发的水分流失。一个标签设置管1至7。 </li><li>解冻10X G7的缓冲区，10X糖蛋白变性缓冲，和10％的NP - 40，并轻轻拍打管混合的内容。保持室温。 </li><li>含酶的样品瓶置于冰上。尽量减少解冻/冻结周期。 </li><li>溶解于600μLDH 2 OhCGβ小瓶（150微克）的内容，并保持在冰上。 </li><li> DH 2 O稀释10倍的股票，准备1毫升的1X G7的缓冲区</li><li> 0.5μLPNGase F稀释25μL1X G7的缓冲区，并保持在冰上。 </li><li>结合α- N - Acetylgalactosaminidase每2μL，α1- 2岩藻糖苷酶，β1- 3半乳糖苷酶和α1- 3，6半乳糖苷酶的准备exoglycosidase组合（如混合）。 </li><li>设置PCR管所示: </li></ol>AK"&gt; <table border="1"><tbody><tr><td> 管/样品＃ </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> hCGβ（0.25mg/ml） </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> - </td><td> - </td><td> - </td></tr><tr><td> 10X糖蛋白变性缓冲</td><td>加入1μl </td><td>加入1μl </td><td>加入1μl </td><td>加入1μl </td><td>加入1μl </td><td>加入1μl </td><td>加入1μl </td></tr><tr><td> DH 2 O </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 9μl </td><td> 9μl </td><td> 9μl </td></tr></tbody></table><ol start="9"><li>第管，轻轻混匀并放置在热循环，盖上盖子，变性的蛋白质，94孵育10分钟° C，4 ° C持有（使用PCR管在热循环，极大地防止蒸发，体积小样本）。 </li><li>删除从热循环管和离心机，以消除任何可见凝结。 </li><li>表示添加下列试剂: </li></ol><table border="1"><tbody><tr><td> 样品＃ </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> 10％NP - 40 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td></tr><tr><td> 10X G7的缓冲区</td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5＆万亩;升</td><td> 2.5μl </td></tr><tr><td> PNGase F 1:50 DIL。 </td><td> - </td><td> 2μL </td><td> - </td><td> - </td><td> 2μL </td><td> - </td><td> - </td></tr><tr><td> Deglycosylation混合</td><td> - </td><td> - </td><td> 2μL </td><td> 2μL </td><td> - </td><td> 2μL </td><td> 2μL </td></tr><tr><td>乙二醇混合</td><td> - </td><td> - </td><td> - </td><td> 2μL </td><td> - </td><td> - </td><td> 2μL </td></tr><tr><td> DH 2 O </td><td>加入10μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td></tr><tr><td> 反应总卷。 </td><td> 25μ升 </td><td> 25μ升 </td><td> 25μ升 </td><td> 25μL &lt;/ TD&gt; <td> 25μ升 </td><td> 25μ升 </td><td> 25μ升 </td></tr></tbody></table><ol start="12"><li>关闭使用新帽（丢弃用过的，因为他们不适合一个孵化周期后）的PCR管。 </li><li>混合管轻轻拍打的4倍，然后旋转内容。 </li><li>放置在热循环，并培育管在37 ° C 4小时，然后冷却至4 ° C的样品</li></ol> 2。 deglycosylated样品的SDS - PAGE <ol><li>准备加入4μL1.25M的DTT 130μL新鲜3X还原SDS上样缓冲液。 </li><li>每个样品中加入12.5μL减少准备3X SDS上样缓冲液。 </li><li>管关闭，新的警帽，并轻轻拍打管混合。 </li><li>孵育94℃，5分钟，然后冷却至4 ° C的热循环管</li><li>装入每个样品30μL和10μL在10-20％Tris -甘氨酸凝胶蛋白标记。除部分3.1样品的其余部分。加载中加入10μlColorPlus预染蛋白Marker。 </li><li> Electrophorese在室温下凝胶在130伏，直至染料前端接近凝胶底部。 </li><li>当凝胶运行完毕后，删除自投凝胶，放置在一个小塑料盒与足够的Coomasie蓝染色覆盖凝胶。 </li><li>凝胶染色，轻轻搅拌1小时。 </li><li> destain解决方案在50毫升的凝胶三次洗为30分钟。 </li><li>使用白色的光透射或扫描仪记录图像。另外，干凝胶可在一帧之间的玻璃纸张。 </li></ol> 3。临- Q翡翠检测糖化蛋白SDS - PAGE凝胶300 <ol><li> 2.1凝胶）的同时，负载在10-20％Tris -甘氨酸凝胶，其余样品。负载10＆万亩; ColorPlus预染蛋白Marker升。 </li><li>虽然凝胶是运行溶解DMF PRO - Q翡翠试剂和准备股票的修复，清洗和氧化性解决方案为300 PRO - Q翡翠染色以下产品与试剂盒提供的手册。 </li><li>电泳完成后，取出凝胶和从投它放置在一个塑料盒。 </li><li>修正加入100毫升的修复方案，并留在室温下轻轻摇动过夜的凝胶。 </li><li> 100毫升的洗涤液10至20分钟，在室温詹蒂莱鼓动清洗凝胶。重复用新鲜的洗涤液洗。 </li><li>轻轻摇动孵育30分钟，在氧化溶液25毫升凝胶氧化碳水化合物。 </li><li>步骤3.5中所述的凝胶洗净。 </li><li>凝胶洗涤准备的Pro - Q翡翠300试剂在步骤3.2至25 ml溶解的解决方案中加入500μL新鲜的PRO - Q 300翡翠染色染色试剂盒中提供的缓冲区。 </li><li>凝胶染色加入25毫升的步骤3.8准备污渍和潜伏在黑暗中轻轻摇动90至120分钟。 </li><li>重复步骤3.5中所描述的两个洗步骤。 </li><li>记录在300nm的紫外透射图像。使用80 kDa的标记标示，这是与临 - Q翡翠绿，紫外线图像重叠到白光图片显示预染阶梯的凝胶。 </li><li>比较Coomasie染色凝胶的Pro - Q翡翠染色凝胶步骤3.11 2.10步的图像。 </li></ol> 4。代表性的成果在蛋白质酶deglycosylation后迁移的变化如图2所示。比较对照样品（A组，泳道1）PNGase F治疗（去除N -糖链，2车道），和Deglycosylation混合（PNGase F，再加上远藤和exoglycosidases 取下 O -聚糖，车道3）。没有进一步减少（4车道）额外糖苷酶消化后的大小。除了大规模的变化，乐队变得更加清晰，为聚糖被删除。 A频段的17 kDa的标记（星号）下运行完全deglycosylatedhCGβ多肽（分子量:16 kDa）的代表。其他乐队可能来自不完全deglycosylation，或从多个身份不明的hCGβ样品中的蛋白质（见1巷）。 5日至7车道（对照组），显示相应的糖苷酶的乐队。 翡翠绿的糖蛋白染色显示面板B.此试剂氧化和污渍的一种蛋白质分子中存在的所有聚糖。因此信号强度下降hCGβ是酶deglycosylated（巷1号至4车道）。泳道3和4的剩余信号，表明聚糖的图案，所使用的酶有抗药性的存在。 4车道使用额外的糖苷酶，删除了一些额外的糖残基:蛋白质的迁移是相同的，但可以看出，在强度略有减少染色。抗糖基是目前在所有的蛋白质种类:翡翠绿（缺席的紫外线图像，括号内），说明他们广泛deglycosylated的某些频带均未检出。额外的数据支持hCGβ不均匀糖化的结论。低频段巷2号（箭头）是淡淡的翡翠的绿色形象，而2巷上带是光明的，表明许多聚糖组仍然存在。这些数据支持这样的结论，在小鼠细胞中表达的重组hCGβ包含多个 glycoforms 9。延长，而这些不同glycoforms由于一些多肽没有收到在每共识网站和/或一些聚糖聚糖的糖基化的内在异质性的人即使在相同的蛋白质不。 <img alt="图1" src="/files/ftp_upload/3749/3749fig1.jpg" /> 图1。典型的分泌或细胞表面的糖蛋白糖基化模式。 <img alt="图2" src="/files/ftp_upload/3749/3749fig2.jpg" /> 图2 SDS - PAGE凝胶酶deglycosylationhCGβ。 A组显示一个Coomasie蓝染色，而B组临- Q翡翠300糖化蛋白的可视化结果。样品编号:1，hCGβ控制; 2，PNGase F消化; 3，Deglycosylation混合消化; 4，Deglycosylation Mix Plus的exoglycosidases消化，样品5至7试剂控制（O型，O型糖苷酶甘油; GNA，β- N Acetylglucosaminidase;加仑/ FUC，β1- 3半乳糖苷酶，α1- 3，6半乳糖苷酶和α1- 2岩藻糖苷酶; GalNA，α- N - Acetylglalactosaminidase;神经，神经氨酸酶; PNGase PNGase F）

<ol>
	<li>Spiro, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+glycosylation:+nature,+distribution,+enzymatic+formation,+and+disease+implications+of+glycopeptide+bonds.">Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds.</a> Glycobiology. 12 (4), 43-56 (2002).</li><li>Ohtsubo, K., Marth, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+in+Cellular+Mechanisms+of+Health+and+Disease.">Glycosylation in Cellular Mechanisms of Health and Disease.</a> Cell. 126, 855-867 (2006).</li><li>Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M., Dwek, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+glycosylation+on+the+biological+function+and+structure+of+human+immunoglobulins.">The impact of glycosylation on the biological function and structure of human immunoglobulins.</a> Annual Review of Immunology. 25, 21-50 (2007).</li><li>Mitra, N., Sinha, S., Ramya, T. N. C., Surolia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-linked+oligosaccharides+as+outfitters+for+glycoprotein+folding,+form+and+function.">N-linked oligosaccharides as outfitters for glycoprotein folding, form and function.</a> Trends in Biochemical Sciences. 31, 156-163 (2006).</li><li>Carlsen, R. B., Bahl, O. P., Swaminathan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chorionic+gonadotropin.+Linear+amino+acid+sequence+of+the+beta+subunit.">Human chorionic gonadotropin. Linear amino acid sequence of the beta subunit.</a> Journal of Biological Chemistry. 248 (19), 6810-6812 (1973).</li><li>Tarentino, A. L., Plummer, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deglycosylation+of+Asparagine-linked+Glycans+by+PNGaseF.">Deglycosylation of Asparagine-linked Glycans by PNGaseF.</a> Trends in Glycoscience and Glycotechnology. 5 (23), 163-170 (1993).</li><li>Tretter, V., Altmann, F., Marz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine+amidase+F+cannot+release+glycans+with+fucose+attached+&#945;1-3+to+the+asparagine-linked+N-acetylglucosamine+residue.">Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached &#945;1-3 to the asparagine-linked N-acetylglucosamine residue.</a> European Journal of Biochemistry. 199, 647-652 (1991).</li><li>Koutsioulis, D., Landry, D., Guthrie, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+endo-&#945;-N-acetylgalactosaminidases+with+broader+substrate+specificity.">Novel endo-&#945;-N-acetylgalactosaminidases with broader substrate specificity.</a> Glycobiology. 18 (10), 799-805 (2008).</li><li>Thakur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+glycoforms+of+the+intact+alpha+subunit+of+recombinant+human+chorionic+gonadotropin+by+high-resolution+capillary+electrophoresis-mass+spectrometry.">Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.</a> Analytical Chemistry. 81 (2), 8900-8907 (2009).</li><li>Park, S., Lee, M. R., Shin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+microarrays+as+powerful+tools+in+studies+of+carbohydrate-mediated+biological+processes.">Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes.</a> Chemical Communications. 37, 4389-4399 (2008).</li><li>Zachara, N. E., Vosseller, K., Hart, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+analysis+of+proteins+modified+by+O-linked+N-acetylglucosamine.">Detection and analysis of proteins modified by O-linked N-acetylglucosamine.</a> Current Protocols in Molecular Biology. 95, 17-17 (2011).</li><li>Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2008).</li></ol>

作者受雇于New England Biolabs公司，从而产生许多本文中使用的试剂。

这里介绍的方法使用酶deglycosylation和SDS - PAGE可以提供有价值的信息有关的蛋白糖基化状态，而聚糖特定试剂方便对数据的解释。此协议的目的是为蛋白质糖基化的初步研究，它特别适用于哺乳动物细胞分泌和膜糖蛋白:在这种情况下选择的酶将专门删除所有的 N -糖链， 或N -糖链加上最常见的糖扩大和形成的 O -糖链的核心。糖苷酶具有额外的优势正在温和，用化学deglycosylation方法相比，保持糖和蛋白质的骨干的完整性。 为了阐明入住率（氨基酸是糖化），糖基化的程度，或确定的精细结构的多聚糖，更先进的技术，如米屁股光谱仪，液相色谱仪或核磁共振是必需的。 由于其简单，在这个协议中的几个步骤可以进行调整，替换，和/或合并，以适应不同的实验需要。然而，为了获得结果，可以清楚地解释重要的是要了解它的长处和局限性。首先，特异性和糖苷酶的纯度是至关重要的:只有良好的特点测试蛋白酶和其他污染活动的酶，应使用。不幸的是，没有标准的单位定义为糖苷酶，根据制造商的规格，用户应确定适当的替代。二，谨慎选择的检测是必要的:A）蛋白染色试剂是有用的，只有在一个相对分子质量的重大转变的deglycosylation结果。并不总是得到这样一个明确的结果如下所示。在其他情况下，我们所看到的异常MIdeglycosylation后格拉逊（远远低于预测的分子量，或更慢迁移） 。这种现象是没有很好地理解，但也可以说， 任何在迁移变化是证据表明，该蛋白已deglycosylated B）糖与抗体检测呈现独特的挑战限制其适用性。它已经很难产生一般反聚糖抗体，它们通常是对一个复杂的糖链的目标，这限制了它们的使用提出。此外，一些单克隆抗聚糖抗体显示不受欢迎的交叉反应 10。彗星）凝集素（糖亲和力内在蛋白）是非常适合对糖的检测，加上他们给聚糖结构上的洞察力。然而，并非所有这些有一个狭窄的特异性，很多都只是部分的特点（这意味着他们可能有未知的亲和力）。因此积极的外源凝集素染色提供指示，而不是地理标志的存在证明，VEN糖D）化学标记试剂盒（基于碘酸氧化糖）的首选方法染色所有的糖蛋白，因此非常适合跟随deglycosylation。 处理未知样品时，这是一个很好的做法，包括糖蛋白控制。胎球蛋白是一个现成的N - 和 O -糖蛋白，绒毛膜促性腺激素（两个亚基）也是一个不错的选择。牛血清白蛋白（BSA）可以用来作为阴性对照。但是，应该指出，一些非糖基化蛋白反应轻微的Pro - Q 300翡翠，特别是在高浓度时使用。非糖基化的分子量标准，如在本议定书中使用的预染蛋白质标记，显示锐利带的优势。不仅是这些蛋白质（80 kDa）的反应到临 - Q翡翠300之一的机会。因此，用户可能要运行的一种糖蛋白标准的阶梯，而不是，如Invitroge糖甘蔗N. 最后，简单的凝胶nucleocytosolic糖蛋白（这是一个单一的O型葡萄糖修改）的检测是可能的使用单克隆抗体，沿β- N - Acetylglucosaminidase 特异性控制11。超越了这项技术的描述是这篇文章的范围，但它应该提到糖苷酶在细胞中存在的许多其他糖蛋白和糖复合物的研究是非常有用的工具。一个所有已知的糖基化形式的综合治疗，可以发现在第二版的"糖生物学基础"，可在NCBI的书柜（书架编号:;:20301239关键词NBK1908）免费在线12。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>人绒毛膜促性腺激素β </td><td>西格玛爱秩序</td><td> C6572 </td><td></td></tr><tr><td> PCR管</td><td> VWR </td><td> 20170-004 </td><td></td></tr><tr><td> PCR扩增热循环仪</td><td>应用生物系统公司</td><td> 4359659 </td><td></td></tr><tr><td> PNGase F </td><td> New England Biolabs公司</td><td> P0704 </td><td>提供与缓冲区</td></tr><tr><td>蛋白质Deglycosylation组合</td><td> New England Biolabs公司</td><td> P6039 </td><td>提供与缓冲区</td></tr><tr><td> α- N - Acetylglalactosaminidase </td><td> New England Biolabs公司</td><td> P0734 </td><td>提供带缓冲</td></tr><tr><td> α1- 2岩藻糖苷酶</td><td> New England Biolabs公司</td><td> P0724 </td><td>提供带缓冲</td></tr><tr><td> α1- 3，6半乳糖苷酶</td><td> New England Biolabs公司</td><td> P0731 </td><td>提供带缓冲</td></tr><tr><td> β1- 3半乳糖苷酶</td><td> New England Biolabs公司</td><td> P0726 </td><td>提供带缓冲</td></tr><tr><td> 10X缓冲G7（500 mM磷酸钠） </td><td> New England Biolabs公司</td><td> --- </td><td>提供与PNGaseF或Degl混合</td></tr><tr><td> 10X糖蛋白变性缓冲液（5％SDS，400毫米数码地面电视） </td><td> New England Biolabs公司</td><td> --- </td><td>提供与PNGaseF或Degl混合</td></tr><tr><td> 10％NP - 40 </td><td> New England Biolabs公司</td><td> --- </td><td>提供与PNGaseF或Degl混合</td></tr><tr><td> 3X SDS上样缓冲液（187.5毫米的Tris - HCl pH值6.8，6％SDS，30％甘油，0.03％溴ophenol蓝色） </td><td> New England Biolabs公司</td><td> B7703 </td><td>提供与1.25M数码地面电视</td></tr><tr><td> ColorPlus预染蛋白Marker </td><td> New England Biolabs公司</td><td> P7709 </td><td></td></tr><tr><td> 10-20％的Tris -甘氨酸Multigel </td><td>丽悦生物有限公司</td><td> DCB - 414893 </td><td></td></tr><tr><td>盒式电​​泳单元</td><td>丽悦生物有限公司</td><td> DCB - 303111 </td><td></td></tr><tr><td>电泳电源的EPS 301 </td><td> GE医疗集团</td><td> 18-1130-01 </td><td></td></tr><tr><td>临- Q翡翠300糖蛋白染色试剂盒</td><td> Invitrogen公司</td><td> P - 21857 </td><td></td></tr><tr><td>艳蓝R </td><td>西格玛爱秩序</td><td> B0149 </td><td></td></tr><tr><td> AlphaImager惠普系统</td><td>细胞生物科技公司</td><td> 92-13823-00 </td><td></td></tr></tbody></table>

identification and characterization of protein glycosylation using specific endo- and exoglycosidases

此外共价连接的糖，糖基化，是一个重要的翻译后修饰的蛋白质，可以显着影响，如细 ​​胞粘附分子贩运，清关，和信号转导1-4的过程。在真核生物中，在分泌途径中最常见的糖基化修改，增加共识，天门冬素剩余物（N -连接）;或在丝氨酸或苏氨酸残基（O型连锁）（图1 ）。在真核生物中是高度保守的N -聚糖合成的起始，而最终产品可以在不同物种间，组织，或蛋白质大大不同。有些聚糖保持未修改"（"高甘露糖N -糖链"），或在高尔基（" 复杂的N -糖链"）进一步处理。更大的多样性是O型聚糖，开始与一个共同的 N -乙酰半乳糖胺（半乳糖胺）残留在动物细胞中，但不同的低等生物 1 。 耳鼻喉科"&gt;蛋白质的糖基化的详细分析本身就是一个领域，需要丰富的资源和专业知识，正确执行。然而，各种可用的酶，删除糖（糖苷）成为可能的糖基化的现状有一个总体思路在一个标准的实验室环境中的蛋白质，在这里我们说明分析模型糖蛋白糖苷酶的使用。重组人绒毛膜促性腺激素β（hCGβ）， 它带有两个N -糖链和四个O型聚糖5，该技术只需要简单仪器和典型的消耗品，它可以很容易地适应多种糖蛋白样品的分析。 几种酶可用于并行研究的一种糖蛋白。 PNGase F是能够去除几乎所有类型的N -连接聚糖6,7。 为 O -糖链，有没有可用的酶可以切割完整的低聚糖从日E蛋白的骨干。相反，O型聚糖修剪exoglycosidases一个短的核心，是那么容易由 O -糖苷酶删除。蛋白质Deglycosylation结构包含PNGase F，O -糖苷酶，神经氨酸酶（唾液酸），β1- 4半乳糖苷酶和β- N - Acetylglucosaminidase。它是用来同时去除N -糖链和一些O型聚糖8。最后，Deglycosylation混合补充的其他exoglycosidases混合物（α- N - Acetylgalactosaminidase，α1- 2岩藻糖苷酶，α1- 3，6半乳糖苷酶，β1- 3半乳糖苷酶），这有助于消除否则抗单糖，可目前在某些O -聚糖。 SDS-PAGE/Coomasie蓝色是用于可视化蛋白质迁移糖苷酶治疗前后的差异。此外，糖特定的染色方法，ProQ翡翠- 300，显示减少信号聚糖是帅客ssively删除。此协议是专为少量的糖蛋白（0.5％到2微克）的分析，虽然酶deglycosylation可以扩大规模，以适应需要较大的蛋白质数量。

Watch this Scientific Journal Video about Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases at JoVE.com

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

使用特定的糖苷酶，以消除通过SDS - PAGE的糖蛋白的糖是一种有价值的的方法来检测蛋白质样品聚糖修改初始糖生物学研究的是一个不错的选择。以下deglycosylation变化可以检测凝胶的流动性的变化或与多糖敏感的试剂染色。

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

identification-characterization-protein-glycosylation-using-specific

Research

JoVE Journal

Biology

36.5K 次观看.  New England Biolabs. 使用特定的糖苷酶，以消除通过SDS - PAGE的糖蛋白的糖是一种有价值的的方法来检测蛋白质样品聚糖修改初始糖生物学研究的是一个不错的选择。以下deglycosylation变化可以检测凝胶的流动性的变化或与多糖敏感的试剂染色。

视频: Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

改善凝胶还原β-消除综合O连接和磺基糖组学质谱

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

科研

化学

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

生物化学

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

定量糖组学和蛋白质组学组合式净化策略

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

鞘糖脂抗原的质谱分析

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

免疫与感染

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, including protein folding, stability, enzymatic protection, and biological recognition. N-glycans attached to glycoproteins (such as lactoferrin, lactadherin, and immunoglobulins) cannot be digested by the host and reach the large intestine, where they are consumed by certain beneficial microbes. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate the gut microbiome's beneficial microorganisms. However, the isolation of these new classes of prebiotics requires novel enzymes. Here, we describe the recombinant production of novel glycosidases from different Bifidobacteria strains (isolated from infants, rabbits, chicken, and bumblebee) for improved N-glycan isolation from glycoproteins. The method presented in this study includes the following steps: molecular cloning of Bifidobacterial genes by an in vivo recombinational cloning strategy, control of transformation success, protein induction, and protein purification.

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Bifidobacteria possess a unique genomic capability for N-glycan cleavage. Recombinantly producing these enzymes would be a promising novel tool to release bioactive N-glycans from glycoprotein-rich substrates such as colostrum.

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, ...

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal infectivity of pathogenic species. Due to this, there is increasing interest in characterizing bacterial glycosylation and a need for high-throughput analytical tools to identify these events. Although bottom-up proteomics readily enables the generation of rich glycopeptide data, the breadth and diversity of glycans observed in prokaryotic species make the identification of bacterial glycosylation events extremely challenging.
Traditionally, the manual determination of glycan compositions within bacterial proteomic datasets made this a largely bespoke analysis restricted to field-specific experts. Recently, open searching-based approaches have emerged as a powerful alternative for the identification of unknown modifications. By analyzing the frequency of unique modifications observed on peptide sequences, open searching techniques allow the identification of common glycans attached to peptides within complex samples. This article presents a streamlined workflow for the interpretation and analysis of glycoproteomic data, demonstrating how open searching techniques can be used to identify bacterial glycopeptides without prior knowledge of the glycan compositions.
Using this approach, glycopeptides within samples can rapidly be identified to understand glycosylation differences. Using Acinetobacter baumannii as a model, these approaches enable the comparison of glycan compositions between strains and the identification of novel glycoproteins. Taken together, this work demonstrates the versatility of open database-searching techniques for the identification of bacterial glycosylation, making the characterization of these highly diverse glycoproteomes easier than ever before.

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model.

基于开放搜索的方法在 鲍曼不动杆菌 O-连锁糖肽鉴定中的应用

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal ...

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1&#8722;3:1) and a longer incubation time (48&#8722;72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

<meta charset="utf8"></head><body>使用糖组学指导的糖蛋白质组学分析结直肠癌组织

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

<meta charset="utf8"></head><body>糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组 

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

摘要

探索更多视频

此视频中的章节

改善凝胶还原β-消除综合O连接和磺基糖组学质谱

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

定量糖组学和蛋白质组学组合式净化策略

鞘糖脂抗原的质谱分析

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

基于开放搜索的方法在鲍曼不动杆菌 O-连锁糖肽鉴定中的应用

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

摘要

探索更多视频

此视频中的章节

改善凝胶还原β-消除综合O连接和磺基糖组学质谱

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

定量糖组学和蛋白质组学组合式净化策略

鞘糖脂抗原的质谱分析

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

基于开放搜索的方法在 鲍曼不动杆菌 O-连锁糖肽鉴定中的应用

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组

基于开放搜索的方法在鲍曼不动杆菌 O-连锁糖肽鉴定中的应用