This work was supported by the College of Liberal Arts and Sciences of Arizona State University in the form of laboratory startup funds to CRB. It was also supported by a grant from Flinn Foundation (Grant No. 1977) and by the National Cancer Institute of the National Institutes of Health under Award Number R33CA191110. JA was supported by the National Institute of General Medical Sciences of the National Institutes of Health Postbaccalalureate Research Education Program (PREP) under award number R25GM071798. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

<ol>
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The authors have nothing to disclose.

在一般情况下，成功地生产部分甲基化的糖醇乙酸酯的（PMAAs）从己糖是充满了很多困难，并且比成功制造N- -acetylhexosamine（HexNAc）PMAAs的更稳健。后面，因为它发挥出在这个过程中的每一步该现象的确切机制是未知的，但必须与本-N-乙酰基（而不是羟基）的独特的化学是唯一相对于己糖HexNAcs。后面，因为它涉及酸水解该现象的机制在别处。43说明总之，对于N -methylacetamido成为酸水解期间正电的能力使得该糖苷键，以酸水解抗性。这就解释如上对于N -acetyllactosamine的分析描述HexNAc相对的低产率的己糖（总HexNAc +己糖信号的11.3％，总而不是50％）。值得注意的是，这种相对升HexNAcs的奥尔产量并不能让他们难以在复杂的生物流体和组织进行检测，排除或从大型，复杂的聚糖（ 如 2,4-人，2,6-人，3,4,6衍生聚糖节点简便检测-Man和3,4- GlcNAc的， 图2）。33由于以何种方式聚糖节点XIC峰面积进行归一化（步骤6.3），只要各个HexNAc的相对产率仍然相对于其他HexNAcs一致（这是这样，看在不同样品之间的不同HexNAcs的相对量表1），有用的信息可以得到。这适用于己糖也是如此。此外，我们以前曾表明，己糖到HexNAcs比例显示一致的定量行为，以及33 -这是通过在每个批次样品质量控制（S）掺入连续监测的现象。 在其中进行全甲基的方式对影响最大整体收益率和可重复性HexNAc PMAAs的。特别是，大应小心完全避免全甲基聚糖暴露至碱性含水条件。在这方面的两个关键步骤是:1）在离开在后面（步骤1.3.13和1.3.14）自旋通过解所有的白色沉淀，和2）一旦被加入到磷酸盐缓冲液自旋通过溶液立即混合（步骤1.3.13和1.3.14）。缓冲，而不是简单的盐溶液被包括在此阶段和随后的液/液萃取步骤，以防止该水溶液的无意碱化。我们怀疑在碱性条件下HexNAcs的甲基化和乙酰化2-氨基的乙酰基可发生水解，导致在降低相关联聚糖氯仿的整体提取效率的更极性的仲胺。 失败全甲基HexNAcs最容易辨认的特征是相对于那些6-半乳糖，3,6-曼色谱峰的4联的GlcNAc（4- GlcNAc的）的强度，和背景的过程中最后一列烘烤（ 图3）的基线强度。当4-GlcNAc的PMAA的绝对丰度低，其归一化相对于所有其它HexNAcs丰度也变低的倾向，以同时增加3,4- GlcNAc的的（最显着）。 旨在优化整体收益率和检测的鲁棒性的一些已经作了修改，因为我们最初发布描述此分析方法33这些变化之一是在综合提取离子色谱（XICs）是标准化的方式:为了简单起见，我们现在除以所有己糖XIC面积总​​和每个单独的己糖XIC的面积;同样地，每个单独的HexNAc XIC的区域由所有HexNAc XIC面积的总和除以。如在表1中 ，总体分析间/帧间分析师reproducibil看出性支持有助于它们各自的己糖或HexNAc信号平均13％的简历到&gt; 1％的聚糖节点。 据我们所知，这是真正的自下而上糖组学，其中聚糖首先分解，然后其成分分析，以构建聚糖组合物的定量样品宽肖像的唯一的化身。这里，代替传统的酶促释放，该全甲基过程非还原性消除（释放）O从它们各自的蛋白质，40而Ñ -连接的聚糖酸水解期间释放33作为补充的方法，以传统的自上而下糖组学联的聚糖，这种方法是能够汇集的利益独特聚糖的功能，如"核心岩藻糖基"，"6唾液酸化"，"平分GlcNAc的"，和"beta 1-6支化"到单个分析信号（参见，4,6- GlcNAc的在图2 6-Gal的，3,4,6-满和2,6-曼节点，respe沉着应对）。在传统的自上而下的方法，特征如这些最终依赖于一个或两个键糖基转移酶33的独特的活动通常分布在几十个完整聚糖，有时是困难的或不可能解决（ 例如 ，3-唾液酸化对6唾液酸化）由于一些完整聚糖群众的退化。此外，这里提出的自下而上的方法已经证明在非侵入检测肺癌初始承诺。33进一步的研究正在进行中，以验证这些初步结果，以及关于检测的其它形式的癌症更多的，尚未发表的结果。 这里所描述的方法的最大的限制是，它是在检测的极限方面的约束，如果是要施加到预分离糖蛋白。基于对个别聚糖标准，未公布的分析没有载体蛋白，定量限FO- [R个别聚糖似乎在于低微克范围内。这些绝不是绝对值低定量限，但这个事实并不相对于所述检测其并非设计为与已经没有必要实现低定量限（至少对于这里所描述的目的和原先预期范围无关紧要在我们以前的出版物33）。其实，人的血浆/血清中含有的糖蛋白10S毫克/毫升的浓度范围内，这意味着血液血浆/血清的分析中，我们只注入〜1/100的最终样品体积和分裂40出41件小数量在GC进样口浪费。如果没有这种实践中，一些聚糖节点可以饱和检测器。可以通过传统的自上而下的MALDI-MS或LC-MS基础的方法产生足够的完整聚糖信号糖蛋白的数量皮摩尔不能用这种方法检测。该方法的进一步细化正在进行补救此限制。

糖脂，糖蛋白，蛋白聚糖，和葡糖胺聚糖构成统称为聚糖复杂的异构碳水化合物的四个主要类。如质膜，多糖包被和细胞外基质和流体的普遍存在和积分分量，聚糖参加等不同生化过程如细胞内吞作用，细胞内运输，细胞运动性，信号转导，分子识别，受体活化，细胞粘附，宿主 - 病原体相互作用，细胞间通讯，免疫监视和免疫反应的启动。1几乎存在于各个领域的生活，被称为糖基转移酶的建立聚糖的聚合物的酶与糖苷水解酶（又称糖苷酶，它分解聚糖）建造，改造串联行动，最终产生敲定聚糖聚合物2。虽然每个糖基转移酶可以在不同的糖结合物进行操作，糖基转移酶通常由特定激活核苷酸的糖供体（ 例如，GDP-岩藻糖）的单糖部分转移到亲核受体的某一类别（ 例如，脂质，多肽，核酸，或生长伪造linkage-和端基异构体特异性糖苷键低聚糖）。据估计，蛋白质（尤其是膜和分泌蛋白）的50％以上是在翻译后糖基化修饰的3-初步组合计算提供了相当大的变异性，多功能性和特异性给予通过糖基化的糖蛋白的欣赏。例如，如果一个多肽基片具有仅10的糖基化位点和每个站点可以与只有3种不同的单糖还原末端的1糖苷键，那么，从理论上说，最后的糖蛋白可以假定3 10 = 59049不同身份。在糖蛋白糖苷键通常与侧链氮Ø形成序列的Asn-X-丝氨酸f中的天冬酰胺残基/苏氨酸（X可以是除脯氨酸外的任何氨基酸），得到N- -glycans 2和侧链羟基丝氨酸和苏氨酸残基，以得到õ-glycans 4。一个细胞的糖组的组成（即它的糖基化产物补码）是唯一的，有限的，因为，除了少数例外，糖基转移酶表现出严格的供体，受体和联动特异性。5重要和丰富的血浆糖蛋白遭受异常的糖基化作为下游的后果异常糖基转移酶表达和活性的由于许多病理情况下，尤其是癌症和炎性疾病。6-24 主要是由于后生因素的影响，糖组是显著更加多样化，动态和复杂的比蛋白质组和转录组25,26虽然哺乳动物基因组的约1％的编码形成，修改和聚糖的组装，在一个非模板驱动方式-一个明显的对比27的糖基化进行到多肽和核酸的生物合成。糖基化酶和诸如环境因素如营养物和前体的可用性的相对数量和活性之间的相互影响，最终决定的性质，速率，和糖基化的程度。5,28胚胎发生（ 例如，测定和分化），细胞活化，和进展通过细胞周期的影响的基因表达（ 即，转录和翻译），并改变提供糖基转移酶的特性和量，其活性是细胞的聚糖谱的直接上游的决定因素。因为（部分的）的增殖，粘合剂，和癌细胞的侵入性质类似于那些普通胚发生细胞中，聚糖的生物合成途径（ 例如，前体积累，失调的表达，aberran具体变化的吨修改，结构截短，或新颖的形成）作为普遍的癌症生物标志物指示的肿瘤形成，进展，迁移和侵袭29各个阶段虽然糖基化是高度复杂的，显然仅在糖基化几改变可以使发生和转移。显然，某些"异常"糖基化产物的确使他们逃避免疫识别和生存在恶劣的血管和转移性环境迁移的需求，有利于癌细胞。28,30,31毫不奇怪，实验表明，破坏或阻止的模式改变基因表达和异常聚糖形成可阻止肿瘤的发生。29尽管如此，生物流体样本（ 例如，尿液，唾液，和血浆或血清）中检测出的异常聚糖可能不是癌症直接指标（或其它疾病），而是下游的微妙而显著成果在免疫系统或以不可预知的器官有害条件量化后果变化。32 虽然它们提供了关于糖组通用的信息，许多分子基于交互的糖组学技术（ 例如，外源凝集素/抗 ​​体阵列和代谢/共价标记）取决于检测整个聚糖结构，并且不提供关于单个聚糖的详细结构信息。形成鲜明对比，质谱（MS）可以帮助识别和量化个别聚糖结构并揭示这种结构的信息作为连接位点到多肽芯。失调的表达或仅一个糖基转移酶的活性可以发起在多个糖基化途径有害分子事件的级联。因为每一个糖基转移酶可能不止一个糖缀合物基板上，并在不同的成长多糖聚合物运作，放松管制的生物合成级联产生disproportionally增加只有一个聚糖产品的量，但几个异构类细胞内或细胞外液异常聚糖。33，然而，这些独特的异常聚糖有时被认为是不切实际的作为生物标志物用于癌症或其它聚糖-情感病症，因为，相对于大池的良好调节聚糖，这些异常聚糖代表一小部分是可能甚至常常由这种高度敏感的技术如质谱仍然检测不到。例如，在细胞内和细胞外体液，广泛蛋白质浓度谱（跨越8个数量级）可以防止由该更丰富的物种掩蔽稀少的糖蛋白的检测。32此外，在确定糖基转移酶活仍然相当实用和理论的挑战，因为许多糖基转移酶在临床生物液体缺席或处于非活动状态体外 。尽管consisten难度TLY检测和定量独具特色的聚糖的超微量，质谱的从业者对用人完好聚糖作为临床指标取得了巨大进步。我们最近开发了一种互补的方法来完整聚糖的分析是，采用GC-MS，便于对所有构成分支点和特定键的单糖（"聚糖节点"），它们一起赋予唯一每个聚糖并且在许多的检测案件直接服务的量化有罪糖基转移酶（S）的相对活性分子的代理人。 自从其首次报道于1958年的直接应用到聚糖分析，气相色谱（GC）已经证明一个功能强大的技术来分析每甲基化的单-和二糖，34确定其anomericity和绝对构型，并用于随后的质谱分析将它们分开。35从1984年到2007年，Ciucanu和他的同事引入并精制2005至2008年间所采用氢氧化钠和碘甲烷使用水和氯仿。35,36固相聚糖全甲基技术，接着全甲基聚糖的液/液萃取，康和同事集成了省时的旋-column方式进入全甲基一步。37,38在2008年，戈茨研究小组设计了一个定量的固相全甲基聚糖分析方法采用基质辅助激光解吸电离（MALDI）质谱分析比较和区分潜在的侵入性和非-invasive乳腺癌细胞;然后39，2009年，戈茨团队结合酶和化学释放技术40切割Ø-glycans从完整的糖蛋白在强碱性固相全甲基方案虽然格茨程序同时促进和全甲基化学释放。 Ø-glycans的，它仅适用于预分离的糖蛋白质。我们在2013年修改的这种技术和通过掺入三氟乙酸适于它为整个未分级的生物流体和均质化的组织样本（TFA）水解，还原和乙酰化的步骤。33，这些额外的步骤也释放从糖脂和N-聚糖从糖蛋白-连接的聚糖，并转换他们进入部分甲基化的糖醇乙酸酯（PMAAs， 图1），其独特的甲基化和乙酰化的图案便于通过GC-MS和唯一地分析表征原始完整聚糖聚合物41（ 图2）中的构成聚糖节点33最终，这过程产生在复杂的生物流体的所有聚糖的复合肖像基于独特的聚糖的功能，如"核心岩藻糖基"，"6唾液酸化"，"平分GlcNAc的"，和"beta 1-6支链"直接的，相对定量 - 每个从单个GC-MS chromatograp衍生HIC高峰。本文介绍的全甲基的进一步优化，乙酰化，分离，并用在相对定量的方式改进沿着清理阶段。

<table>
	<tbody>
		<tr>
			<td>Sodium hydroxide beads&#160;</td>
			<td>367176</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>20-40 mesh, reagent grade 97%</td>
		</tr>
		<tr>
			<td>0.9 mL Spin column</td>
			<td>69705</td>
			<td>Pierce division of ThermoFisher Scientific&#160;</td>
			<td>Includes&#160; plugs and polyethylene frits&#160;</td>
		</tr>
		<tr>
			<td>GC-MS autosampler vial (silanized)*</td>
			<td>C4000-9</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Target DP High Recovery Vial, 1.5 mL, 12 x 32 mm, Includes Teflon-lined pierceable caps</td>
		</tr>
		<tr>
			<td>1.5 mL polypropylene test tubes</td>
			<td>05-402-25</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Snap-cap lid</td>
		</tr>
		<tr>
			<td>2 mL polypropylene test tubes</td>
			<td>05-408-138</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Snap-cap lid</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>D8418</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>BioReagent for molecular biology, reagent grade &#62;99.0%</td>
		</tr>
		<tr>
			<td>Iodomethane&#160;</td>
			<td>I8507</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Contains copper as stabilizer, ReagentPlus 99%</td>
		</tr>
		<tr>
			<td>Acetonitrile&#160;</td>
			<td>A955-4</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Optima LC/MS</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>75002436: Sorvall Legend Micro 17 Centrifuge</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>24 x 1.5/2.0 rotor with ClickSeal biocontainment lid. Rotor catalog number: 75003424</td>
		</tr>
		<tr>
			<td>13 x 100 glass test tube (silanized)*</td>
			<td>53283-800</td>
			<td>VWR</td>
			<td>13 x 100 mm borosilicate glass test tubes with screw-cap finish</td>
		</tr>
		<tr>
			<td>Caps for glass test tubes</td>
			<td>14-930-15D</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Kimble&#8482; Black Phenolic Screw Caps; 13mm-415 GPI thread; PTFE-faced rubber liner.</td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>S7653</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>&#62;99.5% pure</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>4440-08</td>
			<td>Macron Fine Chemicals&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid&#160;</td>
			<td>299537</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>99% purified by redistillation for protein sequencing&#160;</td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>71321</td>
			<td>Fluka Analytical&#160;</td>
			<td>99%</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution&#160;</td>
			<td>320145</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>NH3 content: 28.0 - 30.0%</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>AH230-4</td>
			<td>Honeywell Burdick &#38; Jackson</td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>Acetic acid&#160;</td>
			<td>320099</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>99.70%</td>
		</tr>
		<tr>
			<td>Plastic vacuum desiccator&#160;</td>
			<td>Any model of adequate size</td>
			<td>FoodSaver</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic anhydride&#160;</td>
			<td>539996</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>99.50%</td>
		</tr>
		<tr>
			<td>Dichloromethane&#160;</td>
			<td>D143SK-4</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Stabilized HPLC grade</td>
		</tr>
		<tr>
			<td>Acetone&#160;</td>
			<td>9006-03</td>
			<td>J.T.Baker</td>
			<td>Baker Analyzed&#160;</td>
		</tr>
		<tr>
			<td>Heated evaporation manifold (main unit)</td>
			<td>pi18823</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Thermo Scientific* Reacti-Therm* Heating and Stirring Module; Triple-block Model with Heating and Stirring Function</td>
		</tr>
		<tr>
			<td>Heated evaporation manifold (overhead evaporator)</td>
			<td>pi18826</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>ThermoScientific* Reacti-Vap Evaporator, 27-port; For use with triple-block Reacti-Therm heating module</td>
		</tr>
		<tr>
			<td>Aluminum sample-holder blocks for evaporation manifold</td>
			<td>pi18816</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Block, Aluminum, Reacti-Block S-1, Holds 13mm dia test tubes, 13 holes (14 dia. x 45mm deep)</td>
		</tr>
		<tr>
			<td>Gas chromatograph</td>
			<td>Model A7890</td>
			<td>Agilent&#160;</td>
			<td>Equipped with CTC PAL autosampler</td>
		</tr>
		<tr>
			<td>Mass spectrometer&#160;</td>
			<td>GCT Premier (Time-of-Flight)</td>
			<td>Waters&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Split-mode liner (deactivated / silanized)</td>
			<td>5183-4647</td>
			<td>Agilent</td>
			<td>Containing a small plug of silanized glass wool</td>
		</tr>
		<tr>
			<td>DB-5ms GC column</td>
			<td>122-5532</td>
			<td>Agilent</td>
			<td>30 m x 0.25 mm ID x 0.25 micron film thickness</td>
		</tr>
		<tr>
			<td>Chlorotrimethylsilane</td>
			<td>95541</td>
			<td>Sigma-Aldrich&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vacuum desiccator (for glassware silanization)</td>
			<td>EW-06536-30</td>
			<td>Cole-Parmer</td>
			<td>12&#34; wide; 230 mm plate size</td>
		</tr>
		<tr>
			<td colspan="4">*Glassware silanization is carried out in-house, overnight using chlorotrimethylsilane vapor in a large glass vacuum desiccator.</td>
		</tr>
	</tbody>
</table>

glycan node analysis: a bottom-up approach to glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

共离子流色谱图（TIC）示出相对于在其中两个关键全甲基的步骤被不正确执行的情况下成功全甲基，水解，还原，和人的血浆样品的乙酰化示于图3。 HexNAcs相对于己糖的绝对收益率 : Ñ-acetylhexosamine（HexNAc）部分甲基化的糖醇乙酸酯（PMAAs）往往比己糖产量低。43为了估计相对于己糖HexNAcs的绝对产率，在N- -acetyllactosamine（一个Gal1-4GlcNAc二糖）六10微克样品的水10微升进行分析。为末端半乳糖（叔半乳糖）和4- GlcNAc的TIC峰面积进行了整合。 4- GlcNAc的相对于叔 - 半乳糖和4- GlcNAc的总量的比例为11.3±0.7％（SEM）。虽然HexNAc产量是可再现的（见下文），但事实上，这个值大于0.5的理论值小得多表示HexNAcs的产率比己糖的根本低。 （该HexNAc理论产量为0.5，因为ñ-acetyllactosamine是1:1，己糖HexNAc糖） 区域内和批间重现性 : 内和分析间为贡献的总己糖或HexNAc信号中的至少1％的所有聚糖节点再现性在表1中提供。这些数据是由3个独立的分析上3个独立的天获得的，使用EDTA血浆的相同的库存。人血浆（ 即 ，那些与总己糖或HexNAc信号的&gt; 1％）在图4中所提供的18个最丰富的多糖节点此优化的协议下自动进样器的稳定性数据。 其他值得注意的观察 : 同时优化全甲基方法，我们发现，这是没有必要防止水吸附到初始洗涤，用乙腈现有的氢氧化钠珠。我们还发现，少量水的过程中组合的最后的乙酰化步骤，加热与乙酸酐在短时间内没有TFA的存在有助于促进反应完全。最后，时间飞行（TOF）质谱高分辨力是没有必要的成功聚糖节点分析:基于有关的GC-TOF-MS和传统的传输四极同组样品的平行喷射的初步结果基于GC-MS的选择离子监测工作（SIM）模式展现在最后归己的条款和HexNAc相对丰度相似的结果。 er.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/53961/53961fig1.jpg" /> 图1.全球聚糖甲基化分析过程的分子概述一个O -连接聚糖图示;这些都在全甲基过程，这已被改编为40 Goetz的继全甲基和水解释放，单糖被减少和新生羟基经乙酰化"标记"。甲基化和乙酰化在最终部分甲基化的糖醇乙酸酯（PMAAs）的独特模式对应于原始完整聚合物在独特的"聚糖节点"，并提供了由GC-MS。1.分离和定量-连接的分子基础和糖脂聚糖被释放的酸水解过程中联动标示单糖。从博尔赫斯等人改编。33许可。&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53961/53961fig2.jpg" /> 图2的分析概念的概念的概述。一种上调糖基转移酶（ 例如 ，对GnT-Ⅴ）使在一个特定的，唯一联聚糖的单糖残基的量的增加（在本实施例2,6-连接的甘露糖"节点"）哪位，通过其他糖基转移酶的随后的行动，可以导致形成在低拷贝数的异构整个聚糖结构的混合物各-所有这些都可以是难以检测和常规的方式量化。解析汇集在一起​​"聚糖节点"从在所有的异常聚糖结构提供比任何单一完整聚糖的GnT-V活性的更直接的替代测量。 ， 澳 - - 氮同时测量和脂质链接"GLYC<html>在这里所描述（和最初在别处33）表示一个在概念上的新手段来检测和监控聚糖-情感性疾病如癌症整个生物标本的节点"。从10微升血液的血浆样品显示实际提取离子色谱图。相邻数字在聚糖结构单糖残基表示在该较高残基与低残余的位置。如果没有连锁位置在色谱注释表示的残基可以是在终端位置或者在溶液中（ 例如，葡萄糖）的分类。所有的残基。除了唾液酸通过其1-位向下链路;经由其2-位向下唾液酸链接分割在色谱表明在提取离子色谱变化:己糖残基和m / z 116 + 158对于N M / Z 117 +129 -乙酰基（HexNAc）残基。从博尔赫斯等 33改编的权限。61 / 53961fig2highres.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53961/53961fig3.jpg" /> 图3代表结果。总离子流色谱图（国际信托）对于其中A）中的样品被正确处理相同的人血EDTA血浆样品的聚糖节点分析，B）中的白色残余物在全甲基的解决方案，通过该NaOH纺柱进行成用于液/液萃取随后的混合物，和C），这是通过该NaOH柱纺全甲基溶液加入到磷酸盐缓冲液，但不充分混合，然后加入氯仿液/液萃取的。在图2中提供的传奇。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53961/53961fig3highres.jpg" target="_blank">请点击^ h</a>ERE查看此图的放大版本。 <img alt="图4" src="/files/ftp_upload/53961/53961fig4.jpg" /> 图4.自动进样器的稳定性。自动进样器的稳定性超过48小时的人血浆中18个最丰富的多糖节点。在22小时样品完全干燥并在120微升丙酮重构。数据点的每个集群表示相同样品的四个连续注射。黑线涵盖每个节点聚糖的平均标准值的±15％。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53961/53961fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/53961/53961table1.jpg" width="700" /> 表1.区域内和批间的重现性。数值代表总己糖或全部HexN的CV％交流归个别聚糖节点。贡献的总己糖或HexNAc信号中的至少1％的所有聚糖节点都已列出。数据由3个独立的分析师3个独立的天收购，使用EDTA血浆的股票一样。每批次N = 6个样品。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53961/Table_1-Intra_and_Interassay_precision_summary_results.xlsx">请点击此处下载此表为Excel电子表格。</a>

Watch this Scientific Journal Video about Glycan Node Analysis: A Bottom-up Approach to Glycomics at JoVE.com

Glycan Node Analysis: A Bottom-up Approach to Glycomics

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

聚糖节点分析:自下而上的方法，以糖原组学

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

glycan-node-analysis-a-bottom-up-approach-to-glycomics

Nanyang Technological University

Research

JoVE Journal

Chemistry

10.3K 次观看.  Arizona State University. This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

视频: Glycan Node Analysis: A Bottom-up Approach to Glycomics

A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays.

A guided material screening approach to develop sol-gel derived protein doped microarrays using an emerging pin-printing method of fabrication is described. This methodology is demonstrated through the development of acetylcholinesterase and multikinase microarrays, which are used for cost-effective small-molecule screening.

在导游的带领下材料的筛选方法定量溶胶 - 凝胶蛋白芯片开发

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a ...

科研

化学

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

BMP-2的共价结合利用表面上的自组装单层膜法

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

一个反分析方法化工运输的油漆表征

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

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 ...

Ethylene Polymerizations Using Parallel Pressure Reactors and a Kinetic Analysis of Chain Transfer Polymerization

We demonstrate a method for high-throughput catalyst screening using a parallel pressure reactor starting from the initial synthesis of a nickel &#945;-diimine ethylene polymerization catalyst. Initial polymerizations with the catalyst lead to optimized reaction conditions, including catalyst concentration, ethylene pressure and reaction time. Using gas-uptake data for these reactions, a procedure to calculate the initial rate of propagation (kp) is presented. Using the optimized conditions, the ability of the nickel &#945;-diimine polymerization catalyst to undergo chain transfer with diethylzinc (ZnEt2) during ethylene polymerization was investigated. A procedure to assess the ability of the catalyst to undergo chain transfer (from molecular weight and 13C NMR data), calculate the degree of chain transfer, and calculate chain transfer rates (ke) is presented.

A protocol for the high-throughput analysis of polymerization catalyst, chain transfer polymerizations, polyethylene characterization, and reaction kinetic analysis is presented.

乙烯聚合中使用并联电抗器的压力和链转移聚合的动力学分析

We demonstrate a method for high-throughput catalyst screening using a parallel pressure reactor starting from the initial synthesis of a nickel ...

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 ...

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.

Herein, we describe the fabrication and operation of a double-layer microfluidic system made of polydimethylsiloxane (PDMS). We demonstrate the potential of this device for trapping, directing the coordination pathway of a crystalline molecular material and controlling chemical reactions onto on-chip trapped structures.

微流控气动笼:在芯片水晶捕获，处理的新方法和控制化学处理

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. ...

Original Experimental Approach for Assessing Transport Fuel Stability

The study of fuel oxidation stability is an important issue for the development of future fuels. Diesel and kerosene fuel systems have undergone several technological changes to fulfill environmental and economic requirements. These developments have resulted in increasingly severe operating conditions whose suitability for conventional and alternative fuels needs to be addressed. For example, fatty acid methyl esters (FAMEs) introduced as biodiesel are more prone to oxidation and may lead to deposit formation. Although several methods exist to evaluate fuel stability (induction period, peroxides, acids, and insolubles), no technique allows one to monitor the real-time oxidation mechanism and to measure the formation of oxidation intermediates that may lead to deposit formation. In this article, we developed an advanced oxidation procedure (AOP) based on two existing reactors. This procedure allows the simulation of different oxidation conditions and the monitoring of the oxidation progress by the means of macroscopic parameters, such as total acid number (TAN) and advanced analytical methods like gas chromatography coupled to mass spectrometry (GC-MS) and Fourier Transform Infrared - Attenuated Total Reflection (FTIR-ATR). We successfully applied AOP to gain an in-depth understanding of the oxidation kinetics of a model molecule (methyl oleate) and commercial diesel and biodiesel fuels. These developments represent a key strategy for fuel quality monitoring during logistics and on-board utilization.

Oxidation stability of transport fuels has become a concern for future fuel development. This work presents an original methodology developed by IFP Energies Nouvelles for assessing fuel stability using two different reactors. This methodology was successfully applied to gain an in-depth understanding of the oxidation kinetics and pathways of model molecules and commercial fuels.

评定运输燃料的稳定性原始实验方法

The study of fuel oxidation stability is an important issue for the development of future fuels. Diesel and kerosene fuel systems have undergone several ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

利用毛细管电泳从植物组织量化有机酸:一个测试用例检查<em&gt;小粒种咖啡</em&gt;种子

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

合成阿普塔默-PEI-g-PEG改性金纳米粒子装载多索鲁比辛用于靶向药物输送

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...