This project was supported by funds from Commonwealth Scientific and Research Organisation Flagship Collaborative Research Program, provided to the High Fibre Grains Cluster via the Food Futures Flagship. AB also acknowledges the support of an Australia Research Council (ARC) grant to the ARC Centre of Excellence in Plant Cell Walls (CE110001007).

W-溶胶AXS 1.还原性末端的标签（RE）糖残基与2-氨基苯甲酰胺（2AB） <ol><li>在65℃孵育的W-溶胶AXS用在1M的NaBH 3 CN（氰基硼氢化钠）（pH5.5）中进行2小时的存在2AB（0.2M）到多糖主链链的还原末端转化成它们的荧光衍生物。 注意:以下步骤应在通风橱作为加入NaBH 3 CN释放有毒的氰化物气体，当它是在与水接触来进行。 <ol><li>称出的NaBH 3 CN（62.8毫克）和溶解于水（1毫升）的微量离心管（1.5ml）中以制备1M的加入NaBH 3 CN溶液。在1M的NaBH 3 CN溶液（1ml）中加热在65溶解2AB试剂（27.2毫克） &#160; ℃，调节反应混合物（0.2M 2AB，1M的NaBH 3 CN）与10％乙酸的pH 5.5的pH值。 </li><li>添加反应混合物中加入200μl（0.2M 2AB，1M的NaBH 3 CN）至W-所以升AXS（1毫克）在有盖的玻璃管，混合使用涡旋混合器。孵育在65 2小时&#160;在通风橱°以下。酷悬浮RT，并添加4卷。无水乙醇。 </li><li>放置悬浮于冷藏（4℃）O / N，以沉淀多糖。 </li><li>离心机（1500 XG，10分钟，RT），除去上清液。用无水乙醇（4次），丙酮（1x）和甲醇（1×）广泛洗涤沉淀，每次洗涤之间离心。在40°CO / N真空干燥。 注意:彻底清洗也消除残留2AB。 </li></ol></li></ol> 2.来自2 AB标记的代低聚木糖的W-溶胶AXS <ol><li>溶解2AB在微量离心管的乙酸钠缓冲液500微升（100毫米，pH值5）（1.5毫升）标记的W-溶胶AXS（1毫克）。加入4单位木聚糖内切的（GH 11，[M1]），在37孵育&#160; °下16小时。 </li><li>通过加热反应mixt破坏酶的活性URE在沸水浴10分钟。冷却该悬浮液至室温并转移到玻璃管用帽。添加4卷。无水乙醇，并将该悬浮液在冷藏（4℃）O / N，以沉淀任何未消化的多糖。 </li><li>离心（1500 xg离心10分钟，室温）以分离未消化多糖（片）和木聚糖内切产生低聚木糖（上清液）。倒出上清到一个干净的玻璃管，并把它变成一个温水浴（40℃）。 </li><li>蒸发在氮气流下乙醇到终点体积（〜500微升）。冻结在-80℃的上清液4小时，并在一个冷冻干燥器回收低聚木糖干燥冷冻上清液。 </li></ol> 3.从KOH-溶胶AXS和2AB标签低聚木糖的生成<ol><li>对待切木聚糖酶KOH-溶胶AXS（GH 11，[M1]）以上（部分2.1-2.4）所描述的生成低聚木糖。 </li><li>治疗endoxylanase产生低聚木糖从KOH溶胶AXS以上（部分1.1.1-1.1.2）描述2AB反应混合物（0.2M 2AB，1M硼氢化钠3 CN）。 </li><li>倒出上清到一个干净的玻璃管，并把它变成一个温水浴（40℃）。蒸发在氮气流下乙醇到终点体积（〜500微升）。冻结在-80℃的上清液4小时，并在一个冷冻干燥器回收低聚木糖干燥冷冻上清液。 </li></ol> 4. MALDI-TOF-MS <ol><li> MALDI基质溶液的制备<ol><li>加的2，5- dihydroxbenzoic苯甲酸（DHB）的小舀到含有0.1％甲酸的一管（MALDI-基质溶液）50％乙腈（500毫升）中。混合使用一个旋涡，如果迅速溶解，加DHB的另一个小瓢。注:MALDI-矩阵的理想解决方案浓度为10微克/微升-1。 </li></ol></li><li> MALDI-目标板的制作<ol><li>存款aqueo我们寡糖（本机）的样品（5-10微克）（W-溶胶和/或KOH-SOL）到MALDI-目标板。使用单独的尖端添加0.3微升的MALDI基质溶液和通过上下抽吸混合。使混合物在RT干燥。 注意:正确干燥的样品应当包括向着光点的中心指向长针形晶体。如果存款是粘性和/或油污样品既可以过于集中或由盐，这些沉积物是不可能产生良好的光谱和样本应进一步纯化。 </li><li>引入目标板进入MS源和正（+ IVE）离子模式。调整加速器电压到离子源1 19.0千伏和离子源2 16.3千伏调整激光功率，以大于70％。选择在MALDI-靶板样品当场单击开始，开始激光发射。 注意:目标的所有区域不会产生信号。特定的点只会给一个信号，这或者是由于一些激光发射采样/基质混合物或晶体的特性的枯竭。 <ol><li>移动激光到目标的不同区域采集和平均在大约200随机光谱以获得满意的信号 - 噪声。 </li></ol></li></ol></li></ol> 5. ESI-QTOF-MS <ol><li>采用纳米HPLC加上使用RP电喷雾电离（ESI）飞行（QTOF）MS仪器在线色谱分离的四极时分析切木聚糖酶生成低聚糖（本机）C-18柱（75微米×150毫米; 3.5微米珠尺寸）。 <ol><li>所述木聚糖内切产生的含水寡糖混合物转移到小瓶中，并放入HPLC自动采样器。编程的5-80％在乙腈中，分别在流动相的0.1％（体积/体积）的水甲酸和0.1％（V / V）甲酸的梯度洗脱，经60分钟。 </li><li>调节流速，以0.2微升/分钟。调整正离子模式中的扫描范围帘气体10，GS1 4，源温度100℃，离子喷雾电压2300伏，和去聚类电位50:300-1,600 米/ z和的0.5次/秒使用ESI源条件如下一扫描速率的五，运行LC色谱程序和洗脱低聚糖。所得到的总离子色谱（TIC）是由软件自动保存。 </li><li>打开保存的TIC，并选择提取色谱图。在命令行中键入预期的群众（如 271，403，535，667，799 931 M / Z）。点击进入扫描色谱图。处理，根据制造商的说明14使用软件:ESI-QTOF MS色谱数据的选择离子扫描。 </li></ol></li></ol> 6. ESI- 质谱 <ol><li>插入1〜2微升每-O-甲基化的寡糖在50％乙腈样品放入使用注射器一纳米喷雾尖端（如由Pettolino 等人 1所记载的甲基化）。修剪使用玻璃切割器的纳米喷嘴以适合附连到MS的离散纳米喷雾支架。 </li><li>根据预期的质量范围（200-1,500 米/ z）和帘气体至10，离子喷雾电压1,900 V和极性到正集的质量。 </li><li>按下获取按钮以打开相关的窗口中，输入数据的文件名 ​​，以获得总离子扫描（ESI-MS 1）。然后按STOP按钮。 </li><li>从产品的离子更改扫描类型片段的关注峰。输入感兴趣的质量（ 如885 M / Z）产生碎片，并调整质量范围（200-900 M / Z）。按下获取按钮和调整碰撞能量（在化合物标签向上和/或向下），以实现母离子的整个碎片（885米/ z）和获得的片段离子扫描（ESI-MS 2）。 </li><li>输入感兴趣（ 如711 M / Z）片段离子的质量，并调整质量范围（200-720 米/ z）表示 。按采样按键的d调整碰撞能量，以达到前体离子的整个碎片（710 米/ z）和获得的片段离子扫描（ESI-MS 3）。 </li></ol> 7. H 1 NMR光谱<ol><li>溶解寡糖木聚糖内切生成混合物（KOH-溶胶，天然形式）（〜500微克）在D 2 O中（1.0毫升，99.9％）在一个塑料试管（15毫升）中。冻结在-80℃的悬浮液4小时，在冷冻干燥机以回收低聚木糖干燥冰冻的悬浮液中。 </li><li>为了与D 2 O.充分交流的H 2 o重复两次7.1 </li><li>溶解干燥的寡糖在D 2 O中（0.6ml毫升，99.9％），并添加丙酮的0.5微升（在D 2 O中的5％）作为内标。转移氘化寡糖进入NMR样品管中。 </li><li>持由顶部核磁共振含有管样品，并在塑料离心器插入样品管。放置在采样深度尺微调。脓H或拉该样品管以调节样品的深度，以保证样品的顶部和底部深度计的中心线是相等的。 </li><li>除去深度规和插入所述试样附连到配备有冷冻探针的600MHz的NMR波谱仪自动采样器。 </li><li>登录和开放式光谱仪控制软件。输入样品文件名。打开现有的数据集，然后使用"EDC"命令将其保存到一个新的名称。键入样品的位置一批在自动进样器，然后按"ENTER"。 注:按"ENTER"键会轻轻滴样品管的磁铁孔中，其中，将被定位在探针的顶部。 </li><li>通过在命令行中输入"edte"设置所需的样品温度。等待样品温度继续下一步骤之前达到所需的值。进入"锁定"在命令行，并选择适当的溶剂（D 2 O）。等待＆＃34;锁定完成"的消息显示在窗口的底部。 </li><li>键入"ATMM"在命令行，然后单击"优化"的ATMM菜单栏的顶部。选择启动调谐和探针匹配所选通道（在此情况下1H）。 </li><li>在为垫补过程中，轻微的调整对磁场做出直到均匀磁场通过在命令行中输入"ZG"，进入周围的sample.Acquire实现了信号命令行中键入"topshim"。 </li><li>分析根据制造商的使用在2.225 ppm的参考丙酮的内标1小时的化学位移说明使用软件15光谱。 </li></ol>

<ol>
	<li>Pettolino, F. A., Walsh, C., Fincher, G. B., Bacic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+polysaccharide+composition+of+plant+cell+walls.">Determining the polysaccharide composition of plant cell walls.</a> Nature Protocols. 7, 1590-1607 (2012).</li><li>Ratnayake, S., Beahan, C. T., Callahan, D. L., Bacic, 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+reducing+end+sequence+of+wheat+endosperm+cell+wall+arabinoxylans.">The reducing end sequence of wheat endosperm cell wall arabinoxylans.</a> Carbohydr. Res. 386, 23-32 (2014).</li><li>Bacic, A., Harris, P. J., Stone, B. A., Preiss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biochemistry of Plants, Vol. 14, Carbohydrates. 14, 297-371 (1988).</li><li>York, W. S., O'Neill, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+control+of+xylan+biosynthesis+-+which+end+is+up?.">Biochemical control of xylan biosynthesis - which end is up?.</a> Plant Biol. 11, 258-265 (2008).</li><li>Fincher, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revolutionary+times+in+our+understanding+of+cell+wall+biosynthesis+and+remodeling+in+the+grasses.">Revolutionary times in our understanding of cell wall biosynthesis and remodeling in the grasses.</a> Plant Physiol. 149, 27-37 (2009).</li><li>Faik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylan+Biosynthesis:+News+from+the+Grass.">Xylan Biosynthesis: News from the Grass.</a> Plant Physiol. 153, 396-402 (2010).</li><li>Scheller, H. V., Ulskov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemicelluloses.">Hemicelluloses.</a> Annu. Rev. Plant Biol. 61, 263-289 (2010).</li><li>Bacic, A., Stone, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+(1&#8594;3)-+and+(1&#8594;4)-linked+&#946;-D-glucan+in+the+endosperm+cell-wall+of+wheat.">A (1&#8594;3)- and (1&#8594;4)-linked &#946;-D-glucan in the endosperm cell-wall of wheat.</a> Carbohydr. Res. 82 (13), 372-377 (1980).</li><li>Comino, P., Collins, H., Lahnstein, J., Beahan, C., Gidley, M. J. <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+soluble+and+insoluble+cell+wall+fractions+from+rye,+wheat+and+hull-less+barley+endosperm+flours.">Characterisation of soluble and insoluble cell wall fractions from rye, wheat and hull-less barley endosperm flours.</a> Food Hydrocolloids. 41, 219-226 (2014).</li><li>Pena, M. J., 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=Arabidopsis+irregular+xylem8+and+irregular+xylem9:+Implicationsfor+the+Complexity+of+Glucuronoxylan+Biosynthesis.">Arabidopsis irregular xylem8 and irregular xylem9: Implicationsfor the Complexity of Glucuronoxylan Biosynthesis.</a> Plant Cell. 19, 549-563 (2007).</li><li>Andersson, S. I., Samuelson, O., Ishihara, M., Shimizu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+reducing+end-groups+in+Spruce+xylan.">Structure of the reducing end-groups in Spruce xylan.</a> Carbohydr. Res. 111, 283-288 (1983).</li><li>Mazumder, K., York, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+of+arabinoxylans+isolated+from+ball-milled+switchgrass+biomass.">Structural analysis of arabinoxylans isolated from ball-milled switchgrass biomass.</a> Carbohydr. Res. 345, 2183-2193 (2010).</li><li>Kulkarni, A. 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=The+ability+of+land+plants+to+synthesize+glucuronoxylans+predates+the+evolution+of+tracheophytes.">The ability of land plants to synthesize glucuronoxylans predates the evolution of tracheophytes.</a> Glycobiol. 22 (2012), 439-451 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Agilent MassHunter Workstation Software - Quantitative Analysis Familiarization Guide. , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Topspin User Manual. , (2010).</li><li>Domon, B., Costello, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+nomenclature+for+carbohydrate+fragmentation+in+FAB-MS/MS+spectra+of+glycoconjugates.">A systematic nomenclature for carbohydrate fragmentation in FAB-MS/MS spectra of glycoconjugates.</a> Glycoconjugate. J. 5, 397-409 (1988).</li><li>Hoffmann, R. A., Leeflang, B. R., De Barse, M. M. J., Kamerling, J. P., Vliegenthart, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+by+1H-n.m.r.+spectroscopy+of+oligosaccharides,+derived+from+arabinoxylans+of+white+endosperm+of+wheat,+that+contain+the+elements+----4)[alpha-L-Araf-(1----3)]-beta-D-Xylp-(1----+or+----4)[alpha-+L-Araf-(1----2)][alpha-L-Araf-(1----3)]-beta-D-Xylp-(1----.">Characterisation by 1H-n.m.r. spectroscopy of oligosaccharides, derived from arabinoxylans of white endosperm of wheat, that contain the elements ----4)[alpha-L-Araf-(1----3)]-beta-D-Xylp-(1---- or ----4)[alpha- L-Araf-(1----2)][alpha-L-Araf-(1----3)]-beta-D-Xylp-(1----.</a> Carbohydr. Res. 221, 63-81 (1991).</li><li>Gruppen, H., Hoffmann, R. A., Kormelink, F. J. M., Voragen, A. G. J., Kamerling, J. P., Vliegenthart, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+by+1H+NMR+spectroscopy+of+enzymically+derived+oligosaccharides+from+alkali-extractable+wheat-flour+arabinoxylan.">Characterisation by 1H NMR spectroscopy of enzymically derived oligosaccharides from alkali-extractable wheat-flour arabinoxylan.</a> Carbohydr. Res. 233, 45-64 (1992).</li><li>Kosik, O., Bromley, J. R., Busse-Wicher, M., Zhang, Z., Dupree, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+enzymatic+cleavage+of+cellulose+using+polysaccharide+analysis+by+carbohydrate+gel+electrophoresis+(PACE).">Studies of enzymatic cleavage of cellulose using polysaccharide analysis by carbohydrate gel electrophoresis (PACE).</a> Methods Enzymol. 510, 51-67 (2012).</li></ol>

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大多数基质相细胞壁多糖似乎已经无规取代的主链（有两个糖基和非糖基残基），根据植物物种，发育阶段和组织类型3是高度可变的。由于多糖是次级基因产物的序列没有模板产生的，因此没有单一的分析方法，例如存在对于核酸和蛋白质，它们的测序。纯化的特定键的水解酶的可用性提供了一个有力的工具来降解多糖寡糖然后，可以是色谱分馏，和结合的化学和物理技术使用时完全测?...

Heteroxylans是属于禾本科的主壁和所有被子植物3-6的次生壁的主要非纤维素多糖多糖家族。木聚糖主链中它们的类型和与糖基（葡糖醛酸（中，GlcA），阿拉伯糖（阿拉伯糖））和非糖基（O-乙酰基，阿魏酸），这取决于组织类型，发育阶段和物种7个残基的取代的图案不同。 从小麦壁（ 普通小麦 ）胚乳组成主要由阿拉伯木聚糖（AXS）（70％）和（1→3）（1→4）-β-D-葡聚糖（20％）与少量的纤维素和heteromannans的（2％各）8。木聚糖主链可以是各种未取代的和主要的单取代的（主要是O-2位和在较小程度上O-3的位置）和二 - 取代的（O-2和O-3位）用α-L-阿拉˚F残留9。还原性末端（RE）异质从双子叶植物的木聚糖（例如， 拟南芥 ）10和裸子植物（例如，云杉（ 云杉 ））11包含一个特性四糖的糖基序列; -β-D-木糖对 - （1→3）-α-L-鼠李糖对 - （1→2）-α-D-半乳糖p A-（1→4）-D-的Xyl 页 。理解heteroxylan生物合成和功能（生物和工业），重要的是要充分序列木聚糖主链理解的种类和取代的模式以及还原端（RE）的序列。 用于还原端（RE）和heteroxylans的内部区域的糖基序列（多个）的结构特征的特定的技术在本手稿中描述。该技术依赖于荧光团标记（用2氨基苯甲酰胺（2AB））之前，酶（木聚糖内切）水解heteroxylan链的还原端（RE）。这种方法，特别是用于对RE测序，是首先由纽约实验室10,12-13报道，但现在扩展为包括内部区域测序，并且是已建立的技术的组合是同样适用于独立的隔离的源的所有heteroxylans。这种方法也可以使用（如果有的话），相应的内切水解酶应用于其他类多糖。 在本研究中，如所述脱浆小麦胚乳细胞壁中分离作为不溶醇残基（空气）中，依次用水（W-溶胶FR）萃取，含有1％的NaBH 4（KOH-溶胶FR）1M KOH在Ratnayake 等人。（2014）2。从两个W-和KOH溶胶FRS释放寡糖然后甲基化并且两个天然和甲基化寡糖的详细的结构分析使用MALDI-TOF-MS，ESI-QTOF-MS-加上HPLC用的组合进行使用RP C-18柱在线色谱分离和ESI-MSñ。木聚糖内切消化的KOH溶胶AXS还特征在于核磁共振（NMR）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 aminobenzamide (2AB)</td>
			<td>Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td>A89804</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium borohydride (NaBH4)</td>
			<td>Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td>247677</td>
			<td>Hazardous, handle with care</td>
		</tr>
		<tr>
			<td>sodium cyanoborohydride (NaBH3CN)</td>
			<td>Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td>156159</td>
			<td>Hazardous, handle with care</td>
		</tr>
		<tr>
			<td>endo-1,4-&#946;-Xylanase M1 (from Trichoderma viride) (120101a)</td>
			<td>Megazyme (www.megazyme.com)</td>
			<td>E-XYTR1</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium Oxide (D2O)</td>
			<td>Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td>151882</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze dryer (CHRIST-ALPHA 1-4 LD plus)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RP C18 Zorbax eclipse plus column&#160;</td>
			<td>Agilent&#160;</td>
			<td></td>
			<td>(2.1&#215;100 mm; 1.8 &#181;m bead size)&#160;</td>
		</tr>
		<tr>
			<td>MicroFlex MALDI-TOF MS&#160;&#160; (Model - MicroFlex LR)</td>
			<td>(Bruker Daltonics, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(ESI) -(QTOF) MS&#160;&#160; (Model # 6520)</td>
			<td>(Agilent, Palo Alto, CA )</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ESI-MSn &#160;- ion-trap&#160; (Model # 1100 HCT)</td>
			<td>(Agilent, Palo Alto, CA).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker Avance III 600 MHz -NMR</td>
			<td>Bruker Daltonics, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Topspin (version 3.0)-Biospin- software&#160;</td>
			<td>Bruker&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GC-MS (Model # 7890B)</td>
			<td>Agilent&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

sequencing of plant wall heteroxylans using enzymic, chemical (methylation) and physical (mass spectrometry, nuclear magnetic resonance) techniques

这个协议描述了用于还原末端（RE）和heteroxylans的内部区域的糖基序列（多个）的表征的具体的技术。脱浆小麦胚乳细胞壁中分离作为不溶醇残基（AIR）1，并依次用水（W-溶胶星期五）萃取和含有1M的KOH 1％的NaBH 4（KOH溶胶星期五），为通过Ratnayake 等描述人 （2014）2。两种不同的方法（ 见图1中的摘要）被采用。在第一，完整的W-溶胶AXS用2AB处理来标记原始RE主链糖残基，然后用木聚糖内切处理，以生成2AB标记的RE和内部区域分别还原的低聚糖，的混合物。在第二种方法中，所述KOH溶胶星期五与内木聚糖酶水解成第一生成其随后用2AB标记的寡糖的混合物。该酶解释放（（UN）的标签）从两个低聚糖W-和KOH溶胶FRS然后甲基化并且两个天然和甲基化寡糖的详细的结构分析使用MALDI-TOF-MS，RP-HPLC-ESI-QTOF-MS和ESI-MS n的组合被执行。木聚糖内切消化的KOH溶胶AXS的特征还在于用核磁共振（NMR）也提供了关于异头配置信息。这些技术可以应用于其他类使用适当的内切水解酶多糖。

的木聚糖内切消化2AB标记的W-溶胶AXS生成2AB标记的RE寡糖的混合物和一系列从木聚糖链（ 图1中的内部区域衍生的未标记的（无2AB标签）寡糖;从Ratnayake 等人 。 2）。然后将一系列的色谱方法被用来分馏异构体的复杂混合物。最后，MS技术被用于识别异构结构，然后由质谱技术测序。这里，我们提出的代表，而不是该方法的全面的，例子。...

Watch this Scientific Journal Video about Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques at JoVE.com

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

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Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Principles of Nuclear Magnetic Resonance

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7.6K 次观看.  University of Melbourne. This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR). 

视频: Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

使用氢交换质谱分析蛋白质动力学

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

科研

化学

Laser-heating and Radiance Spectrometry for the Study of Nuclear Materials in Conditions Simulating a Nuclear Power Plant Accident

Major and severe accidents have occurred three times in nuclear power plants (NPPs), at Three Mile Island (USA, 1979), Chernobyl (former USSR, 1986) and Fukushima (Japan, 2011). Research on the causes, dynamics, and consequences of these mishaps has been performed in a few laboratories worldwide in the last three decades. Common goals of such research activities are: the prevention of these kinds of accidents, both in existing and potential new nuclear power plants; the minimization of their eventual consequences; and ultimately, a full understanding of the real risks connected with NPPs. At the European Commission Joint Research Centre&#39;s Institute for Transuranium Elements, a laser-heating and fast radiance spectro-pyrometry facility is used for the laboratory simulation, on a small scale, of NPP core meltdown, the most common type of severe accident (SA) that can occur in a nuclear reactor as a consequence of a failure of the cooling system. This simulation tool permits fast and effective high-temperature measurements on real nuclear materials, such as plutonium and minor actinide-containing fission fuel samples. In this respect, and in its capability to produce large amount of data concerning materials under extreme conditions, the current experimental approach is certainly unique. For current and future concepts of NPP, example results are presented on the melting behavior of some different types of nuclear fuels: uranium-plutonium oxides, carbides, and nitrides. Results on the high-temperature interaction of oxide fuels with containment materials are also briefly shown.

We present experiments in which real nuclear fuel, cladding, and containment materials are laser heated to temperatures beyond 3,000 K while their behavior is studied by radiance spectroscopy and thermal analysis. These experiments simulate, on a laboratory scale, the formation of a lava-phase following a nuclear reactor core meltdown.

核材料在核电厂事故模拟条件下的激光加热和辐射光谱研究

Major and severe accidents have occurred three times in nuclear power plants (NPPs), at Three Mile Island (USA, 1979), Chernobyl (former USSR, 1986) and ...

Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to the gas phase, and efficiently transferred to a detection system. Fizzy extraction takes advantage of the effervescence phenomenon. First, a carrier gas (here, carbon dioxide) is dissolved in the sample by applying overpressure and stirring the sample. Second, the sample chamber is decompressed abruptly. Decompression leads to the formation of numerous carrier gas bubbles in the sample liquid. These bubbles assist the release of the dissolved analyte species from the liquid to the gas phase. The released analytes are immediately transferred to the atmospheric pressure chemical ionization interface of a triple quadrupole mass spectrometer. The ionizable analyte species give rise to mass spectrometric signals in the time domain. Because the release of the analyte species occurs over short periods of time (a few seconds), the temporal signals have high amplitudes and high signal-to-noise ratios. The amplitudes and areas of the temporal peaks can then be correlated with concentrations of the analytes in the liquid samples subjected to fizzy extraction, which enables quantitative analysis. The advantages of fizzy extraction include: simplicity, speed, and limited use of chemicals (solvents).

Fizzy extraction is a new laboratory technique for analysis of volatile and semivolatile compounds. A carrier gas is dissolved in the liquid sample by applying overpressure and stirring the sample. The sample chamber is then decompressed. The analyte species are liberated to the gas phase due to effervescence.

挥发性有机化合物与大气压化学电离四极杆质谱联用

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

利用二次 Nanoelectrospray 电离耦合高分辨质谱的实时呼吸分析

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

寡 peptoids 的合成及质谱分析

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

单金属纳米粒子的高分辨率物理特性

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

采用多段注射-毛细管电泳质谱法进行高通量和综合药物监测

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

用于测定金属结合寡聚肽的金属电子识别和机制的Ion流动性-质谱技术

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy represents an important technique to understand the structure and bonding environments of molecules. There exists a drive to characterize materials under conditions relevant to the chemical process of interest. To address this, in situ high-temperature, high-pressure MAS NMR methods have been developed to enable the observation of chemical interactions over a range of pressures (vacuum to several hundred bar) and temperatures (well below 0 &#176;C to 250 &#176;C). Further, the chemical identity of the samples can be comprised of solids, liquids, and gases or mixtures of the three. The method incorporates all-zirconia NMR rotors (sample holder for MAS NMR) which can be sealed using a threaded cap to compress an O-ring. This rotor exhibits great chemical resistance, temperature compatibility, low NMR background, and can withstand high pressures. These combined factors enable it to be utilized in a wide range of system combinations, which in turn permit its use in diverse fields as carbon sequestration, catalysis, material science, geochemistry, and biology. The flexibility of this technique makes it an attractive option for scientists from numerous disciplines.

The molecular structures and dynamics of solids, liquids, gases, and mixtures are of critical interest to diverse scientific fields. High-temperature, high-pressure in situ MAS NMR enables detection of the chemical environment of constituents in mixed phase systems under tightly controlled chemical environments.

高温高压原位魔角旋转核磁共振光谱

Nuclear magnetic resonance (NMR) spectroscopy represents an important technique to understand the structure and bonding environments of molecules. There ...

Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor

In-cell NMR is a unique approach to observe the structural and dynamic properties of biological macromolecules at atomic resolution directly in living cells. Protein folding, chemical modifications, and conformational changes induced by ligand binding can be observed. Therefore, this method has great potential in the context of drug development. However, the short lifetime of human cells confined in the NMR spectrometer limits the application range of in-cell NMR. To overcome this issue, NMR bioreactors are employed that can greatly improve the cell sample stability over time and, importantly, enable the real-time recording of in-cell NMR spectra. In this way, the evolution of processes such as ligand penetration and binding to the intracellular protein target can be monitored in real time. Bioreactors are often limited by low cell viability at high cell numbers, which results in a trade-off between the overall sensitivity of the experiment and cell viability. We recently reported an NMR bioreactor that maintains a high number of human cells metabolically active for extended periods of time, up to 72 h. This setup was applied to monitor protein-ligand interactions and protein chemical modification. We also introduced a workflow for quantitative analysis of the real-time NMR data, based on multivariate curve resolution. The method provides concentration profiles of the chemical species present in the cells as a function of time, which can be further analyzed to obtain relevant kinetic parameters. Here we provide a detailed description of the NMR bioreactor setup and its application to monitoring protein-ligand interactions in human cells.

This protocol describes the setup of an NMR bioreactor to keep encapsulated human cells viable for up to 72 h, followed by time-resolved in-cell NMR data acquisition and analysis. The methodology is applied to monitor intracellular protein-ligand interactions in real time.

使用高细胞密度生物反应器通过实时定量细胞内 NMR 监测人细胞中蛋白质-配体相互作用

In-cell NMR is a unique approach to observe the structural and dynamic properties of biological macromolecules at atomic resolution directly in living ...