这项工作得到了中国自然科学基金的部分支持 (批准号31471703、A0201300537 和31671854至合资企业和李涅、赠款 31470435 G.Y.) 和100外国人才计划 (批准编号 JSB2014012 合资企业)。

1. 样品收集
<ol><li>购买不同品种的鲜萝卜 (萝卜)。</li>
</ol>2. 从萝卜中分离蛋白质
<ol><li>融汇约100克新鲜萝卜与厨房搅拌机10分钟。</li>
	<li>将浆料转移到50毫升离心管和离心机在 1.4万 x克4 摄氏度, 以消除不溶性的材料。</li>
	<li>将上清液小心地转移到新的50毫升离心管中, 加入等量的2米乙酸酸 (TCA) 溶液。 注: 添加 TCA 将沉淀可溶性 (糖) 蛋白。</li>
	<li>离心机在 1.4万 x g在4°c 为30分钟并且从颗粒 (糖) 蛋白质去除上清。</li>
	<li>用20毫升去离子水和离心机清洗颗粒, 1.4万 x克4 摄氏度, 去除可溶性寡糖和多糖。</li>
	<li>重复步骤2.5。四次。</li>
	<li>在1毫升的去离子水中重新悬浮小球, 转移到新鲜的1.5 毫升离心管。</li>
</ol>3. n-多糖的制备
<ol><li>从步骤2.7 中混合50µL 蛋白质溶液。(等于5克新鲜萝卜), 0.2 亩重组 PNGase H+ 10 毫米醋酸。</li>
	<li>孵育反应混合物在37°c 为12小时。孵化后, 离心机在 1.4万 x g 5 分钟内除去多余的蛋白质和酶。</li>
	<li>将上清液转移到新鲜的1.5 毫升离心管。</li>
</ol>4. n-多糖的纯化
<ol><li>制备固相萃取 (SPE) 柱, 以丰富n-多糖, 从反应混合物中除去盐和其他杂质, 提高荧光 2-苯酰胺 (2-AB) 标记剂对n-糖的选择性衍生。<ol>
<li>加入3毫升的去离子水洗涤柱。</li>
		<li>加入3毫升80% 乙腈溶液, 含三氟乙酸酸 (TFA, 0.1% 伏/v) 活化 SPE 柱。</li>
		<li>加入3毫升去离子水, 平衡 SPE 柱。</li>
	</ol>
</li>
	<li>将该示例从步骤3.3 转移到该列, 然后丢弃该流程。</li>
	<li>加入1.5 毫升的去离子水洗涤柱并丢弃流经。</li>
	<li>洗脱从柱上释放的n-多糖, 使用1.5 毫升水中20% 乙腈溶液, 含有 0.1% TFA (v/v) 到2毫升离心管中。</li>
	<li>室温离心蒸发去除溶剂。蒸发, 直到样品完全干燥。</li>
</ol>5. n-多糖的荧光衍生化
<ol><li>准备1毫升的 2 ab 溶液, 包括35毫米 2 AB 和0.1 米 cyanoborohydride 钠在二甲基亚砜/乙酸溶液 (7:3, v/v)。</li>
	<li>添加5µL 2 AB 溶液的干燥样品 (步骤 4.5) 来源于六不同的萝卜。漩涡每个样品直到完全地溶化。</li>
	<li>在65摄氏度孵育2小时的混合物。</li>
	<li>让样品冷却到室温5分钟, 加入5µL 去离子水和40µL 乙腈。离心管在 1.4万 x g 3 分钟。</li>
	<li>将48µL 从上清到300µL 高回收率 HPLC 瓶。将衍生物糖样品存放在摄氏-20 摄氏度, 可达一个月。</li>
</ol>6. HILIC-UPLC 多糖的分析
<ol><li>使用与在线荧光 detectorset 连接的标准 UPLC 系统, 分别在 330 nm/420 nm 的激发/发射波长上分析样品。</li>
	<li>使用疏水性相互作用液相色谱 (HILIC)-UPLC 糖柱在60°c 的柱温度为分析。</li>
	<li>通过稀释50毫升的库存溶液 (1 米甲酸铵溶液, pH 4.5), 用950毫升的液相色谱-质谱 (LCMS) 级水, 制备溶剂 A。准备库存解决方案, 如下所示:<ol>
<li>添加43毫升甲酸到700毫升的 LCMS 级 H2O。</li>
		<li>用滴状加水氨溶液 (25% 瓦特/瓦) 将 pH 值调整为4.5。</li>
		<li>将溶剂转移到测量缸内, 并填充1000毫升, LCMS 级 H2o 型存储在4-6 摄氏度, 可达3月。</li>
	</ol>
</li>
	<li>采用 LCMS 级乙腈为溶剂 B。</li>
	<li>用以下梯度洗脱分离n-多糖:<ol>
<li>从95% 的溶剂 B 开始加入溶剂 a. 将流量设置为0.5 毫升/分, 从0到44.5 分钟。</li>
		<li>从0分钟到6分钟, 逐渐减少溶剂 B 与线性梯度 to78 的比例。</li>
		<li>从6分钟到44.5 分钟, 逐渐降低溶剂 B 的比例, 线性梯度为55.9%。</li>
		<li>从44.5 分钟到46.5 分钟, 快速将溶剂 B 的比例从55.9% 降到 0%, 并在0% 处保持2分钟, 并开始洗涤柱。将流量降低到0.25 毫升/分, 从44.5 到55分钟。</li>
		<li>通过将溶剂 B 从48.5 分钟增加到50.5 分钟, 并在95% 处保持4.5 分钟, 进一步洗涤柱。</li>
		<li>将该柱重新平衡95% 溶剂 B 的起始条件为0.5 毫升/分, 从50.5 到57分钟。</li>
	</ol>
</li>
	<li>在 UPLC 系统中注入45µL 的样品。</li>
	<li>洗脱多糖由 UPLC 与保留时间在15和40分钟之间。</li>
	<li>用2毫升离心管收集每个观测到的 UPLC 峰值分数, 并通过离心蒸发干燥样品。</li>
	<li>注射约 1 pmol 2 AB 标记葡聚糖标准 (2−20葡萄糖单位), 以校准植物n-糖的分析, 并分配他们的洗脱时间为标准化的葡萄糖单位。</li>
</ol>7. MALDI 分析
<ol><li>在5µL LCMS 级水中溶解6.8 步的样品。混合1µL 的样品溶液和1µL 6-aza 2 thiothymine (的) 溶液 (0.3% 瓦特/v 在70% 伏/五水中乙腈), 并将混合物移到 MALDI-飞行样品载体上。</li>
	<li>通过按下启动按钮, 在正离子模式下使用 MALDI 质谱仪对样品进行分析。</li>
	<li>采用随附的 MALDI 分析软件对质谱进行分析。</li>
	<li>使用开放源码糖解释软件19解释频谱。设置 2 AB 标记的搜索参数, [M + Na]+, 并将精度参数设置为 2.0 Da。</li>
</ol>

<ol>
	<li>Altmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Protein+Glycosylation+in+Allergy.">The Role of Protein Glycosylation in Allergy.</a> International Archives of Allergy and Immunology. 142 (2), 99-115 (2007).</li><li>Van Ree, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;+(1,+2)-xylose+and+&#945;+(1,+3)-fucose+residues+have+a+strong+contribution+in+IgE+binding+to+plant+glycoallergens.">&#946; (1, 2)-xylose and &#945; (1, 3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens.</a> Journal of Biological Chemistry. 275 (15), 11451-11458 (2000).</li><li>Biswas, H., Chattopadhyaya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+Curcuma+longa+rhizome+lectin:+Role+of+N-linked+glycosylation.">Stability of Curcuma longa rhizome lectin: Role of N-linked glycosylation.</a> Glycobiology. 26 (4), 410-426 (2016).</li><li>Lefebvre, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+N-glycosylation+sites+and+CXC+motifs+in+trafficking+of+Medicago+truncatula+Nod+factor+perception+protein+to+plasma+membrane.">Role of N-glycosylation sites and CXC motifs in trafficking of Medicago truncatula Nod factor perception protein to plasma membrane.</a> Journal of Biological Chemistry. 287 (14), 10812-10823 (2012).</li><li>Severino, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+glycan+moiety+on+the+structure-function+relationships+of+PD-L1,+type+1+ribosome-inactivating+protein+from+P.+dioica+leaves.">The role of the glycan moiety on the structure-function relationships of PD-L1, type 1 ribosome-inactivating protein from P. dioica leaves.</a> Molecular BioSystems. 6 (3), 570-579 (2010).</li><li>Lige, B., Ma, S., Van Huystee, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+the+site-directed+removal+of+N-glycosylation+from+cationic+peanut+peroxidase+on+its+function.">The effects of the site-directed removal of N-glycosylation from cationic peanut peroxidase on its function.</a> Archives of Biochemistry and Biophysics. 386 (1), 17-24 (2001).</li><li>Ceriotti, A., Duranti, M., Bollini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+N-glycosylation+on+the+folding+and+structure+of+plant+proteins.">Effects of N-glycosylation on the folding and structure of plant proteins.</a> Journal of Experimental Botany. 49 (324), 1091-1103 (1998).</li><li>Kamerling, J. P., Gerwig, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+the+Structural+Analysis+of+Carbohydrates.">Strategies for the Structural Analysis of Carbohydrates.</a> Comprehensive Glycoscience. , 1-68 (2007).</li><li>Huang, Y., Mechref, Y., Novotny, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+nonreductive+release+of+O-linked+glycans+for+subsequent+analysis+through+MALDI+mass+spectrometry+and+capillary+electrophoresis.">Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis.</a> Analytical Chemistry. 73 (24), 6063-6069 (2001).</li><li>Triguero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+enzymatic+N-glycan+release+comparison+for+N-glycan+profiling+of+monoclonal+antibodies+expressed+in+plants.">Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants.</a> Analytical Biochemistry. 400 (2), 173-183 (2010).</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+&#8594;+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 &#8594; 3 to the asparagine-linked N-acetylglucosamine residue.</a> European Journal of Biochemistry. 199 (3), 647-652 (1991).</li><li>Fan, J. -. Q., Lee, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+studies+on+substrate+structure+requirements+of+glycoamidases+A+and+F.">Detailed studies on substrate structure requirements of glycoamidases A and F.</a> Journal of Biological Chemistry. 272 (43), 27058-27064 (1997).</li><li>Altmann, F., Paschinger, K., Dalik, T., Vorauer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine+amidase+A+and+its+N-glycans.">Characterisation of peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine amidase A and its N-glycans.</a> European Journal of Biochemistry. 252 (1), 118-123 (1998).</li><li>Altmann, F., Schweiszer, S., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+comparison+of+peptide:+N-glycosidases+F+and+A+reveals+several+differences+in+substrate+specificity.">Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity.</a> Glycoconjugate Journal. 12 (1), 84-93 (1995).</li><li>Wang, T., Voglmeir, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PNGases+as+valuable+tools+in+glycoprotein+analysis.">PNGases as valuable tools in glycoprotein analysis.</a> Protein and Peptide Letters. 21 (10), 976-985 (2014).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+characterization+of+a+novel+extremely+acidic+bacterial+N-glycanase+with+combined+advantages+of+PNGase+F+and+A.">Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A.</a> Bioscience Reports. 34 (6), 00149 (2014).</li><li>Du, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+sample+preparation+methodology+for+plant+N-.glycan+analysis+using+acid-stable+PNGase+H+.">Rapid sample preparation methodology for plant N-.glycan analysis using acid-stable PNGase H+.</a> Journal of Agricultural and Food Chemistry. 63 (48), 10550-10555 (2015).</li><li>Wang, T., Hu, X. -. C., Cai, Z. -. P., Voglmeir, J., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+Quantitative+Analysis+of+Carbohydrate+Modification+on+Glycoproteins+from+seeds+of+Ginkgo+biloba.">Qualitative and Quantitative Analysis of Carbohydrate Modification on Glycoproteins from seeds of Ginkgo biloba.</a> Journal of Agricultural and Food Chemistry. 65 (35), 7669-7679 (2017).</li><li>Ceroni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GlycoWorkbench:+a+tool+for+the+computer-assisted+annotation+of+mass+spectra+of+glycans.">GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans.</a> Journal of Proteome Research. 7 (4), 1650-1659 (2008).</li></ol>

作者没有什么可透露的。

我们在这里提出的协议允许比较不同品种的萝卜的n-糖剖面。与现有协议相比, 这种方法的一个显著优点是, 多糖的酶释放与 2 AB 的衍生反应之间没有缓冲的变化。这个过程最关键的步骤是用 SPE 柱纯化n-多糖, 因为在反应混合物中除去盐或其他杂质可能会对荧光衍生效率产生负面影响。此处提出的方法仅允许对不同的n-多糖的相对丰度进行评估;绝对量化需要在步骤2.1 中添...

近年来, 植物中的n-多糖引起了越来越多的关注, 因为先前的研究突出了n-多糖作为免疫交叉反应的潜在来源, 可能引发过敏反应1,2.此前已证明, 植物糖蛋白的n-多糖会影响催化活性3,4, 热稳定性和折叠5,6或亚细胞定位和分泌物7。为了使糖结构与它们各自的功能相关联, 多糖必须首先从化学或酶的糖蛋白中释放出来。经典的化学方法释放n-和O型多糖是β消除, 其中碱性样品处理是伴随着减少与硼氢化, 以产生一个糖醇8。然而, 这一过程排除了荧光的标记, 并导致单糖单位从糖结构的减少端显著衰减。基于氢氧化铵/碳酸盐处理的化学 deglycosylation 也是常用的替代方法9。这两种化学释放方法都没有降解完整的蛋白质, 这允许在不存在同质量范围内的肽片段干扰的情况下对未标记的糖池进行质谱分析。然而, 这些方法的一个缺点是增加的降解率α1,3-fucosylated n-多糖, 一个共同的碳水化合物结构发现在植物10。另外, 使用肽的酶释放方法:n-glycanases (PNGases, EC 3.5.1.52) 也被广泛应用。重组 PNGase F (从杆菌 meningosepticum) 是最常见的选择, 允许释放所有类型的n-多糖, 除了轴承核心α1,3 岩藻糖11,12的结构。因此, PNGase (从杏仁籽中分离) 通常用于植物n-多糖13的分析。然而, 这种酶 deglycosylates 只 proteolytically 衍生糖肽, 无法 deglycosylate 本族糖蛋白14。因此, 在进一步分析之前需要进行多步骤的抽样检查, 从而导致大量的多糖损失, 尤其是低丰度的15。该方法的总体目标是以简单、稳健的方式为n-糖发布和荧光标记提供一个优化的工作流。基本的基本原理是, 最近在Terriglobus 长春发现的 PNGase H+可以 recombinantly 表达在大肠杆菌中, 可以直接从蛋白质支架中水解n-多糖酸性情况16。使用 PNGase H+替代方法的一个关键优点是荧光标记反应可以在同一反应管内进行, 而不会改变反应缓冲器17,18。低丰寡糖的制备条件简单, 回收率高, 使该方法成为多糖分析的重要工具. 该协议适用于不同植物种类的n-多糖分析。

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="880">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Chemicals:</td>
			<td style="width:123px;"></td>
			<td style="width:207px;"></td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichloroacetic acid&#160;</td>
			<td>SCR, Shanghai</td>
			<td>80132618</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid glacial</td>
			<td>Huada, Guangzhou</td>
			<td>64-19-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>General-reagent</td>
			<td>G80988C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trifluoroacetic acid</td>
			<td>Energy chemical</td>
			<td>W810031</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-aminobenzamide</td>
			<td>Heowns, Tianjin</td>
			<td>A41900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium cyanoborohydride</td>
			<td>J&#38;K Scientific Ltd</td>
			<td>314162</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide</td>
			<td>Huada, Guangzhou</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2AB-labeled dextran ladder, 200 pmol</td>
			<td>Agilent Technologies</td>
			<td>AT-5190-6998</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-Aza-2-thiothymine&#160;</td>
			<td>Sigma</td>
			<td>275514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tools/Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kitchen blender</td>
			<td>Bear, Guangzhou</td>
			<td>LLJ-A10T1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Techcomp</td>
			<td>CT15RT</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifugal Evaporator</td>
			<td>Hualida, Taicang</td>
			<td>LNG-T120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SPE column</td>
			<td>Supelco</td>
			<td>Supelclean ENVI Carb SPE column</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MALDI-TOF mass spectrometer</td>
			<td>Bruker</td>
			<td>Autoflex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HPLC Analysis:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High-recovery HPLC vial</td>
			<td>Agilent Technologies</td>
			<td>&#160;# 5188-2788</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC System</td>
			<td>Shimadzu</td>
			<td>Nexera</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Detector for HPLC</td>
			<td>Shimadzu</td>
			<td>RF-20Axs&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Column oven</td>
			<td>Hengxin</td>
			<td>CO-2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Column</td>
			<td>Waters</td>
			<td>Acquity UPLC BEH glycan column</td>
			<td>2.1 &#215; 150 mm, 1.7 &#956;m particle size</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Water</td>
			<td>Merck Millipore</td>
			<td>#WX00011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Acetonitrile</td>
			<td>Merck Millipore</td>
			<td># 100029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Aladdin</td>
			<td>F112034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonia solution</td>
			<td>Aladdin</td>
			<td>A112080</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of n-glycans from raphanus sativus cultivars using pngase h+

近年来, 植物的碳水化合物基团受到了相当大的关注, 因为它们是交叉反应, 引起过敏性免疫反应的潜在来源。此外, 碳水化合物结构在植物代谢中也起着至关重要的作用。在这里, 我们提出了一个简单和快速的方法, 以制备和分析不同品种的萝卜 (萝卜) 的n-多糖, 利用n-glycanase 的具体释放植物衍生碳水化合物结构。为了达到这一目的, 萝卜组织匀浆的粗乙酸酸沉淀处理 PNGase H+, 并标记使用 2-苯酰胺作为荧光标记。随后用超效液相色谱 (UPLC) 分离法和基质辅助激光解吸电离-飞行时间 (MALDI) 质谱法对糖样品进行了分析, 详细结构对萝卜衍生n-糖结构的相对 abundancies 进行评价和量化。该协议也可用于对不同植物种类的n-多糖进行分析, 对多糖对人体健康的作用和作用的进一步研究有一定的参考价值。

图 1显示了描述的协议的示意图, 包括从萝卜中分离 (糖) 蛋白、 n-多糖的制备、UPLC 分析以及这些成分的 MALDI--MS 分析。图 2显示了萝卜品种衍生物多糖的代表性 UPLC 图谱。图 3显示了2衍生物n-糖结构用 MALDI-飞行质谱法获得的结果。图 4显示了每个萝卜品种的n-</e...

Watch this Scientific Journal Video about Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+ at JoVE.com

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

萝卜品种多糖的 PNGase 研究

本文介绍了一种简便、快速的方法, 对不同品种萝卜 (萝卜) 的n-多糖进行了制备和分析。

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, ...

analysis-of-n-glycans-from-raphanus-sativus-cultivars-using-pngase-h

Research

JoVE Journal

Biochemistry

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

6.5K 次观看.  Nanjing Agricultural University. 本文介绍了一种简便、快速的方法, 对不同品种萝卜 (萝卜) 的n-多糖进行了制备和分析。

视频: Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

激光显微切割RNA测序实验设计:从玉米叶片发展的经验分析

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

科研

生物化学

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

从多个小鼠组织分离中间丝状蛋白以研究与老化相关的翻译后修饰

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

人视网膜解剖与 RPE-脉络膜的蛋白质组分析

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

用最小组织量测定果蝇透纤维中线粒体耗氧量

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2&#8217;-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

生物技术环境下的氟黏膜化人乳三糖--用基因编码生物传感器分析

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

从高梁叶组织中分离出希石，用于自上而下质量光谱分析潜在的表观遗传标记

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.

Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

使用支撑浮块在透射电子显微镜中制备样品支撑膜

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...