Name: synthesis and mass spectrometry analysis of oligo-peptoids
Uploaded: 2018-02-21T13:00:00.000+00:00
Duration: 11 min 44 s
Description: Watch this Scientific Journal Video about Synthesis and Mass Spectrometry Analysis of Oligo-peptoids at JoVE.com

作者要感谢 Zuckermann 先生和罗纳德博士 (分子铸造, 劳伦斯伯克利国家实验室) 在 peptoid 合成技术支持。我们感谢国家科学基金会 (CHE-1301505) 的支持。所有质谱实验都是在太平洋大学化学质谱仪的实验室进行的。

1. Peptoid 的合成
注: 合成始于激活树脂, 通过肿胀树脂和去除保护组。接着, 通过重复单体加法循环将 peptoid 链添加到树脂上。第一个与树脂耦合的单体是 C 端残渣。peptoid 从 C 总站拉长到 N 终点。一旦达到所需的 peptoid 序列, 树脂就会脱落, peptoid 产品被提纯。
<ol><li>试剂的制备 注: 用微测量液体试剂, 用分析天平测量固体试剂。<ol>
<li>混合6.2 毫升的 n, n-'-diisopropylcarbodiimide (DIC) 和43.8 毫升的 n, n-甲基酰胺 (dmf), 以准备一个0.8 米的 DIC/DMF 溶液。</li>
		<li>将5.56 克的50.0 毫升的 dmf 分解为0.8 米/dmf 溶液。</li>
		<li>混合2毫升哌啶 (Pip) 和8毫升的 dmf, 以达到 20% Pip/dmf 解决方案。</li>
		<li>将1.9 毫升的北角溶解为13.1 毫升的 dmf, 以达到1.0 米北角/dmf。</li>
		<li>将1.3 毫升的 Nme 分解为13.7 毫升 dmf, 以达到1.0 米 Nme/dmf。</li>
		<li>将0.32 毫升的 Nae 分解为2毫升的 dmf, 以达到1.0 米 Nae/dmf。 注意: 合成中使用的大多数化学物质都是有害的。三氟乙酸酸 (TFA)、DIC、Pip 和医学会对皮肤、眼睛和呼吸道都有危害。DMF 和二氯甲烷 (DCM) 是可疑的致癌物质。所有反应都应在通风罩中进行, 并应使用适当的个人防护设备。请检查合成中使用的所有化学品的材料安全数据表 (MSDS)。</li>
	</ol></li>
	<li>树脂活化
	<ol>
<li>测量出84毫克的溜冰场酰胺树脂 (树脂, 0.047 毫摩尔, 装载0.56 毫摩尔/克), 并将其添加到10毫升聚丙烯固相反应容器。将柱塞插入容器中。</li>
		<li>在反应容器中加入2毫升 DMF, 并将容器盖上压力帽。将容器放在振动筛上, 在室温下以大约12度和385振荡/分钟的运动角度搅动容器, 30 分钟. 通过去除瓶盖并推送反应容器的柱塞, 将溶液排出废物容器。</li>
		<li>将2毫升的 20% Pip/DMF 溶液添加到容器中, 并盖上容器。在振动筛上搅动2分钟, 然后将溶液排出废物容器。</li>
		<li>将2毫升的 20% Pip/DMF 溶液添加到容器中, 将容器盖上, 并在室温下搅动12分钟. 卸下瓶盖, 将溶液排出废物容器。</li>
		<li>通过加入1毫升的 DMF, 将容器盖上, 并将该容器搅拌1分钟, 将树脂洗净. 卸下瓶盖, 然后通过推送活塞来排出溶液。用 DMF 清洗树脂4次。</li>
	</ol></li>
	<li>单体添加和 n-端乙酰化 注: 每个单体添加周期涉及两个反应步骤, bromoacetylation 和位移。<ol>
<li>进行第一单体添加周期, 形成 Nme 树脂。</li>
		<li>执行 bromoacetylation 反应。在烧杯中混合1毫升0.8 米/dmf 溶液和1毫升0.8 米 DIC/dmf 溶液。将混合物转移到含有树脂的反应容器上, 并盖上容器。将容器放在振动筛上, 在室温下搅动20分钟. 卸下瓶盖并将解决方案排放到废物容器。</li>
		<li>通过加入1毫升的 DMF, 将容器盖上, 搅拌1分钟, 并通过推柱塞来排出溶液, 以洗涤树脂。通过加入1毫升的 DCM 来洗涤树脂, 将容器搅拌1分钟, 并排出溶液。再用 DCM 洗涤树脂, 然后用 DMF 洗两次。</li>
		<li>执行位移反应。加入1毫升的1.0 米 Nme/DMF 溶液, 盖上容器, 在室温下搅动容器60分钟. 卸下瓶盖, 然后按下活塞排出溶液。</li>
		<li>通过添加1毫升 DMF, 将该容器搅拌1分钟, 并通过推柱塞来排出溶液, 从而清洗树脂。用1毫升的 DCM 来洗涤树脂, 搅拌1分钟, 并排出溶液。再洗一次, 然后用 DMF 洗两次。</li>
		<li>重复单体添加周期从步骤1.3.2 到1.3.5 形成 Nae-(北 Nme)4-树脂。重复位移反应步骤 (1.3.4) 时, 应根据 peptoid 序列使用特定的胺溶液。 注: peptoid 链从 c 终点延伸至 N 端, 并与树脂的 c 末端残留物结合。</li>
		<li>进行 n-端乙酰化, 形成交 Nae-(Nme)4-树脂。</li>
		<li>将92µL 醋酸酐, 43.5 µL 的 n, diisopropylethylamine (DIPEA), 和2毫升的 DMF 在烧杯中, 使约2毫升乙酰化鸡尾酒。</li>
		<li>在含有树脂的容器中加入2毫升乙酰化鸡尾酒, 将容器盖上, 并在室温下搅动60分钟. 卸下瓶盖, 并通过推送活塞来排出溶液。</li>
		<li>通过加入1毫升的 DMF 来洗涤树脂, 搅拌容器1分钟, 并通过推柱塞来排出溶液。通过加入1毫升的 DCM 来洗涤树脂, 将容器搅拌1分钟, 并排出溶液。再次用 DCM 再次洗涤树脂, 再用 DMF 清洗两次。</li>
		<li>通过加入1毫升的 DCM 来洗涤树脂, 将容器搅拌1分钟, 并通过推柱塞来排出溶液。用 DCM 重复清洗两次。取下瓶盖, 让树脂在反应釜内风干10分钟。</li>
	</ol></li>
	<li>裂解纯化
	<ol>
<li>混合3.8 毫升的 TFA, 100 µL 的 triisopropylsilane (提示), 100 µL 的 HPLC 级 H2O 在烧杯中, 使4毫升的裂解鸡尾酒。</li>
		<li>在装有树脂的容器中加入 4 毫升新鲜的鸡尾酒 , 将容器盖上 , 并在室温下搅动 2 小时。</li>
		<li>取下瓶盖, 将滤液溶液收集到50毫升聚丙烯离心管中。在船上加1毫升的 TFA, 盖上, 搅拌1分钟. 将滤液溶液收集到同一离心管中。</li>
		<li>在氮气气流中轻轻吹 TFA 蒸发, 直到大约1毫升粘性溶液被留下。</li>
		<li>将15毫升乙醚添加到剩余溶液中, 将离心管盖上, 并将其孵化在-20 摄氏度冷藏柜中, 2 小时至一夜。粗 peptoid 沉淀成白色固体。</li>
		<li>用离心机在 4427 x g 10 分钟内将固体颗粒取出. 在不失去固体的情况下, 将离心管的瓶盖和醒酒乙醚放入烧杯中。 注意: 乙醚是一种易燃的有机溶剂。请使用乙基醚安全离心机。</li>
		<li>将10毫升的冰冷二乙基醚放入含有固体的离心管中, 将管盖上, 并将其置于离心机中, 以洗涤固体。进行离心 4427 x g 10 分钟. 将离心机管从离心机中取出, 并将二乙基醚醒酒到烧杯中, 而不会失去固体。</li>
		<li>在氮气流中轻轻吹干固体。</li>
		<li>添加10毫升的 HPLC 级 H2O 溶解干固体。通过尼龙注射器过滤器, 其孔径为0.45 µm, 并将滤液收集到预加权50毫升聚丙烯离心管中。</li>
		<li>壳体通过放置和旋转含有 peptoid 溶液的离心管在12盎司, 双层膨胀聚苯乙烯杯1/3 填充液态氮气, 冻结溶液。Lyophilize 冻结溶液过夜, 以产生固体 peptoid。</li>
		<li>重复冻干一次, 将固体 peptoid 溶解成10毫升的 HPLC 级 H2O, 在液氮中冻结壳, 冻干过夜。结果 peptoid 是足够纯的序列分析的质谱。</li>
	</ol></li>
</ol>2. MS 测量和序列分析
注: ms/毫秒实验是在三极质谱仪上进行的, 耦合到电喷雾电离 (ESI) 源。数据收集是通过使用数据采集软件与仪器相结合来控制的。一般程序包括 1) 进行全扫描质谱实验, 记录质谱, 2) 执行 CID ms/ms 实验, 记录 ms/ms 频谱, 3) 比较 ms/ms 谱数据与理论基于 peptoid 的结构特征, 对破碎方案进行了预测。
<ol><li>样品溶液的制备
	<ol>
<li>在 3-5 毫升玻璃瓶中称量出 1.0-2.0 毫克固体 peptoid。加入1毫升的乙腈和水混合溶剂 (ACN/H2O, 1:1, v/v) 以溶解 peptoid。这给出了一个集中约 10-3 m 的股票样本解决方案。</li>
		<li>将20µL 的库存溶液转移到1.5 毫升离心管中, 加入1毫升混合溶剂 ACN/H2O, 以产生约 10-5 m 的稀释样溶液。</li>
		<li>在稀释样品溶液中去除任何可能不溶的微粒, 在 4427 x g 处进行离心处理3分钟. 将溶液顶部的700µL 转移到另1.5 毫升离心管中, 使 peptoid MS 工作溶液与浓度约 10-5 m。</li>
		<li>在质谱测量中, 根据观察到的信号强度调整 MS 工作溶液的浓度。</li>
		<li>在稀释样品溶液中去除任何可能不溶性微粒的另一种方法是通过0.20 µm 注射器过滤器通过样品溶液, 并将滤液收集到1.5 毫升离心管中, 使 peptoid MS 工作溶液约 10-5m。</li>
	</ol></li>
	<li>记录质谱
	<ol>
<li>运行 ACN/H 的混合溶剂2O (1:1, v/v) 通过电喷雾电离 (ESI) 源, 并建立了一个标准的正离子, 全扫描质谱模式与质量-电荷比 (m/z) 范围 100-1500 的仪器。 注意: 典型操作参数: ESI 针电压, 5 伏 毛细管电压, 40 V 干燥气体 (氮气) 温度, 200 °c。</li>
		<li>将大约300µL peptoid MS 工作溶液 (约 10-5 M) 添加到500µL 或1毫升注射器中, 并使用毛细管聚醚醚酮 (PEEK) 油管将注射器连接到 ESI 入口。将注射器放在注射器泵上, 并将流速设置为10µL/分钟, 将样品溶液注入到 ESI 入口。</li>
		<li>打开 esi 针电压激活 esi 过程, 然后打开探测器。设置在配置文件模式下的显示和 m/z 范围的 100-1500。查看在配置文件窗口中显示的质量频谱配置文件。在 m/z 1265 (显示为 m/z 1264.6) 的峰值对应于 peptoid 离子或质子 peptoid。</li>
		<li>记录 MS 频谱2分钟。在 "方法窗口" 中, 使用2分钟作为 "运行时间"。打开录制窗口并填写正确的文件名, 并开始记录频谱。 注意: 生成的质量频谱显示在图 3中。</li>
		<li>在1265的 m/z 处优化峰值强度。将 m-/z 范围设置为 1150-1350, 并在查看配置窗口中显示的 mV 峰值强度时调整毛细管电压。例如, 将毛细管电压增加到50伏或更高, 并查看峰值强度的变化。最佳峰值强度约为 150-200 mV。 注: 调节毛细管电压可显著改变某些离子的丰度。增加毛细管电压可以提高 peptoid 离子的强度。然而, 高毛细管电压也可能导致离子源中 peptoid 离子的离解, 降低观测强度。调整干燥气体的温度可以提高1265米/z 峰值的强度, 但比调节毛细管电压效果低。如果峰值在 m/z 1265 不达到最佳强度, 调整样品浓度 (或例子, 加倍的浓度的 MS 工作溶液)。</li>
		<li>将仪器切换到 ms/ms 模式。将前体离子设置为 m/z 1265 和 ms/毫秒质量范围在 m/z 100-1400。在 "方法窗口" 中, 使用 m/z 1265 作为 "Q1 第一质量", 并保留 "Q1 最后一个质量" 空白。使用 m/z 100 作为 "Q3 第一质量" 和 m/z 1400 作为 "Q3 最后马萨诸塞州" 注: 在 ms/毫秒模式中, 第一四极单元 (Q1) 作为一种质量过滤器, 用于将 peptoid 离子分离为前体离子, 第二四极单元 (Q2) 为碰撞单元, 第三四极单元 (Q3) 作为质量分析仪。</li>
		<li>设置碰撞能量在 40 eV 和碰撞气体 (在这种情况下, 氩) 压力在 1.5 mTorr。</li>
		<li>查看在配置文件窗口中显示的质量频谱配置文件。在1265的 m/z 的峰值对应于 peptoid 离子, 而具有较低 m-/z 值的峰代表 peptoid 离子的碎片。</li>
		<li>调整碰撞能量, 优化碎片谱的显示。例如, 将碰撞能量增加到 45 eV, 并查看频谱剖面的变化。 注: 一般情况下, 增加碰撞能量会提高碎片离子的丰度, 减少 peptoid 离子的丰度。增加碰撞气体压力也会增加碎片离子的丰度。 注意: 不要增加 2 mTorr 以上的碰撞气体压力。</li>
		<li>记录毫秒/ms 频谱2分钟。在 "方法窗口" 中, 使用2分钟作为 "运行时间"。打开录制窗口并填写正确的文件名, 并开始记录频谱。</li>
		<li>重复录制 1-2 次。</li>
	</ol></li>
	<li>Peptoid 序列分析 注: 在 CID 条件下, peptoid 离子会在 peptoid 主干上的酰胺键上碎裂, 产生一系列称为 B 离子的 N 端片段, 以及一系列称为 Y 离子的 C 末端片段。<ol>
<li>绘制出乙酰 peptoid 的化学结构, 方法是将 N 终点放在左侧和右侧的 C 总站上, 并在每个酰胺键上画一条虚线, 以生成一个碎片方案, 如图 2a所示。用虚圆画一个质子来指示电荷载体。从结构的左侧开始, 将标签放在虚线上, 以指示 N 个终端片段为 b1, b2, 到 b8。从右侧开始, 将标签放在虚线上, 以指示 C 终端片段为 y1、y2、y8。 注: 可利用化学绘图软件绘制 peptoid 结构。</li>
		<li>设想从左侧的 4th酰胺键中进行碎片, 然后用适当的正式电荷绘制 N 端片段和 C 终端片段的结构, 如图 2b所示。将 B4的标签放在 N 终端片段上, 然后将 Y5的标签放在 C 终端片段上。计算 B4的 m-/z 值, 通过汇总结构中元素的标称质量以产生580的值, 并将 m/z 580 放在 N 终端片段上。计算 Y5的 m-/z 值, 并将 m/z 685 放在 C 终端片段上。</li>
		<li>计算所有八 B 离子和所有八个 Y 离子的 m-/z 值, 并将它们组合在一个表中, 如表 1所示。 注意: 碎片的 m-/z 值可以使用化学绘图软件计算。</li>
		<li>使用数据审阅软件与仪器一起打开 peptoid 记录的 ms/ms 频谱。将标签首选项设置为 "显示离子标签" 和标签阈值为3%。这将生成一个在峰值上标有 m/z 值的 ms/毫秒频谱。</li>
		<li>将 ms/ms 光谱数据导出为 CSV 格式的文本文件, 并使用数据处理软件重建频谱。将 m/z 值放在峰值上。生成的 ms/ms 频谱显示在图 4中。 注: 一些质谱仪配备了能够生成 ms/ms 频谱的数据处理软件。在这种情况下, 不需要导出 ms/ms 光谱数据。</li>
		<li>将 ms/MS 频谱上显示的 m-/z 值分配给表 1中显示的数据, 以标识相应的 B 和 Y 离子。例如, m/z 580 的峰值被标识为 B4-离子和 m/z 685 被标识为 Y5-离子。在频谱上用相应的 B 和 Y 符号标记峰值, 如图 4所示。</li>
	</ol></li>
</ol>

<ol>
	<li>Sun, J., Zuckermann, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptoid+Polymers:+A+Highly+Designable+Bioinspired+Material.">Peptoid Polymers: A Highly Designable Bioinspired Material.</a> ACS Nano. 7 (6), 4715-4732 (2013).</li><li>Fowler, S. A., Blackwell, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+in+peptoids:+Recent+advances+toward+deciphering+the+structural+requirements+for+biological+function.">Structure-function relationships in peptoids: Recent advances toward deciphering the structural requirements for biological function.</a> Org. Biomol. Chem. 7 (8), 1508-1524 (2009).</li><li>Chongsiriwatana, N. P., Patch, J. A., Czyzewski, A. M., Dohm, M. T., Ivankin, A., Gidalevitz, D., Zuckermann, R. N., Barron, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptoids+that+mimic+the+structure,+function,+and+mechanism+of+helical+antimicrobial+peptides.">Peptoids that mimic the structure, function, and mechanism of helical antimicrobial peptides.</a> Proc. Natl. Acad. Sci. U. S. A. 105 (8), 2794-2799 (2008).</li><li>Kruijtzer, J. A., Nijenhuis, W. A., Wanders, N., Gispen, W. H., Liskamp, R. M., Adan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptoid-Peptide+Hybrids+as+Potent+Novel+Melanocortin+Receptor.">Peptoid-Peptide Hybrids as Potent Novel Melanocortin Receptor.</a> J. Med. Chem. 48 (13), 4224-4230 (2005).</li><li>Liu, B., Alluri, P. G., Yu, P., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Potent+Transactivation+Domain+Mimic+with+Activity+in+Living+Cells.">A Potent Transactivation Domain Mimic with Activity in Living Cells.</a> J. Am. Chem. Soc. 127 (23), 8254-8255 (2005).</li><li>Patch, J. A., Barron, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helical+Peptoid+Mimics+of+Magainin-2+Amide.">Helical Peptoid Mimics of Magainin-2 Amide.</a> J. Am. Chem. Soc. 125 (40), 12092-12093 (2003).</li><li>Ham, H. O., Park, S. H., Kurutz, J. W., Szleifer, I. G., Messersmith, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifouling+Glycocalyx-Mimetic+Peptoids.">Antifouling Glycocalyx-Mimetic Peptoids.</a> J. Am. Chem. Soc. 135 (35), 13015-13022 (2013).</li><li>Olivier, G. K., Cho, A., Sanii, B., Connolly, M. D., Tran, H., Zuckermann, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-Mimetic+Peptoid+Nanosheets+for+Molecular+Recognition.">Antibody-Mimetic Peptoid Nanosheets for Molecular Recognition.</a> ACS Nano. 7 (10), 9276-9286 (2013).</li><li>Olsen, C. A., Ziegler, H. L., Nielsen, H. M., Frimodt-Moeller, N., Jaroszewski, J. W., Franzyk, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial,+Hemolytic,+and+Cytotoxic+Activities+of+&#946;-Peptoid-Peptide+Hybrid+Oligomers:+Improved+Properties+Compared+to+Natural+AMPs.">Antimicrobial, Hemolytic, and Cytotoxic Activities of &#946;-Peptoid-Peptide Hybrid Oligomers: Improved Properties Compared to Natural AMPs.</a> ChemBioChem. 11 (10), 1356-1360 (2010).</li><li>Lutz, J. -. F., Ouchi, M., Liu, D. R., Sawamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-Controlled+Polymers.">Sequence-Controlled Polymers.</a> Science. 341 (6146), 628 (2013).</li><li>Altuntas, E., Schubert, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Polymeromics":+Mass+spectrometry+based+strategies+in+polymer+science+toward+complete+sequencing+approaches:+A+review.">"Polymeromics": Mass spectrometry based strategies in polymer science toward complete sequencing approaches: A review.</a> Anal. Chim. Acta. 808, 56-69 (2014).</li><li>Paulick, M. G., Hart, K. M., Brinner, K. M., Tjandra, M., Charych, D. H., Zuckermann, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavable+Hydrophilic+Linker+for+One-Bead-One-Compound+Sequencing+of+Oligomer+Libraries+by+Tandem+Mass+Spectrometry.">Cleavable Hydrophilic Linker for One-Bead-One-Compound Sequencing of Oligomer Libraries by Tandem Mass Spectrometry.</a> J. Comb. Chem. 8 (3), 417-426 (2006).</li><li>Thakkar, A., Cohen, A. S., Connolly, M. D., Zuckermann, R. N., Pei, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Sequencing+of+Peptoids+and+Peptide-Peptoid+Hybrids+by+Partial+Edman+Degradation+and+Mass+Spectrometry.">High-Throughput Sequencing of Peptoids and Peptide-Peptoid Hybrids by Partial Edman Degradation and Mass Spectrometry.</a> J. Comb. Chem. 11 (2), 294-302 (2009).</li><li>Sarma, B. K., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submonomer+Synthesis+of+A+Hybrid+Peptoid-Azapeptoid+Library.">Submonomer Synthesis of A Hybrid Peptoid-Azapeptoid Library.</a> ACS Comb Sci. 14 (10), 558-564 (2012).</li><li>Li, X., Guo, L., Casiano-Maldonado, M., Zhang, D., Wesdemiotis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-Down+Multidimensional+Mass+Spectrometry+Methods+for+Synthetic+Polymer+Analysis.">Top-Down Multidimensional Mass Spectrometry Methods for Synthetic Polymer Analysis.</a> Macromolecules. 44 (12), 4555-4564 (2011).</li><li>Figliozzi, G. M., Goldsmith, R., Ng, S. C., Banville, S. C., Zuckermann, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+N-substituted+glycine+peptoid+libraries.">Synthesis of N-substituted glycine peptoid libraries.</a> Methods Enzymol. 267, 437-447 (1996).</li><li>Zuckermann, R. N., Kerr, J. M., Kent, S. B. H., Moos, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+method+for+the+preparation+of+peptoids+[oligo(N-substituted+glycines)]+by+submonomer+solid-phase+synthesis.">Efficient method for the preparation of peptoids [oligo(N-substituted glycines)] by submonomer solid-phase synthesis.</a> J. Am. Chem. Soc. 114 (26), 10646-10647 (1992).</li><li>Utku, Y., Rohatgi, A., Yoo, B., Kirshenbaum, K., Zuckermann, R. N., Pohl, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Multistep+Synthesis+of+a+Bioactive+Peptidomimetic+Oligomer+for+the+Undergraduate+Laboratory.">Rapid Multistep Synthesis of a Bioactive Peptidomimetic Oligomer for the Undergraduate Laboratory.</a> J. Chem. Educ. 87 (6), 637-639 (2010).</li><li>Tran, H., Gael, S. L., Connolly, M. D., Zuckermann, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-phase+submonomer+synthesis+of+peptoid+Polymers+and+their+self-assembly+into+highly-ordered+nanosheets.">Solid-phase submonomer synthesis of peptoid Polymers and their self-assembly into highly-ordered nanosheets.</a> J. Visualized Exp. (57), e3373 (2011).</li><li>Bolt, H. L., Cobb, S. L., Denny, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Efficient+Method+for+the+Synthesis+of+Peptoids+with+Mixed+Lysine-type/Arginine-type+Monomers+and+Evaluation+of+Their+Anti-leishmanial+Activity.">An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity.</a> J Vis Exp. (117), (2016).</li><li>Morishetti, K. K., Russell, S. C., Zhao, X., Robinson, D. B., Ren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+mass+spectrometry+studies+of+protonated+and+alkali+metalated+peptoids:+Enhanced+sequence+coverage+by+metal+cation+addition.">Tandem mass spectrometry studies of protonated and alkali metalated peptoids: Enhanced sequence coverage by metal cation addition.</a> Int. J. Mass Spectrom. 308 (1), 98-108 (2011).</li><li>Bogdanov, B., Zhao, X., Robinson, D. B., Ren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Capture+Dissociation+Studies+of+the+Fragmentation+Patterns+of+Doubly+Protonated+and+Mixed+Protonated-Sodiated+Peptoids.">Electron Capture Dissociation Studies of the Fragmentation Patterns of Doubly Protonated and Mixed Protonated-Sodiated Peptoids.</a> J. Am. Soc. Mass Spectrom. 25 (7), 1202-1216 (2014).</li><li>Ren, J., Tian, Y., Hossain, E., Connolly, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragmentation+Patterns+and+Mechanisms+of+Singly+and+Doubly+Protonated+Peptoids+Studied+by+Collision+Induced+Dissociation.">Fragmentation Patterns and Mechanisms of Singly and Doubly Protonated Peptoids Studied by Collision Induced Dissociation.</a> J. Am. Soc. Mass Spectrom. 27 (4), 646-661 (2016).</li></ol>

作者没有什么可透露的。

使用所介绍的协议合成了一个 nonamer peptoid、交流 Nae (北 Nme)4-NH2。该合成装置涉及一种类似于注射器的聚丙烯固相反应容器和机械振动筛。反应容器是商业上可利用和低成本。机械振动筛是化学实验室中常见的仪器。使用类似于注射器的反应容器, 可以通过手动移动柱塞将溶液抽出并推出容器。该技术允许单体添加和树脂裂解反应发生在一个单一的反应容器中, 并消除了将树脂绑定...

Peptoids 是一类序列控制聚合物, 其骨干结构模仿肽的结构。Peptoids 可以从不同的胺合成, 这使 Peptoids 能够表现出高度可调谐的属性1,2。Peptoids 已被用作生物物理研究的分子模型, 被认为是治疗剂, 并被设计为蛋白质的配体3,4,5,6。Peptoids 已发展成为各种生物活性化合物, 如防污和抗体模拟材料, 抗菌剂, 和酶抑制剂7,8,9。具有高度有序和可调谐的性质, peptoids 也被认为是一种类型的分子信息存储10。这些不同应用的发现要求开发有效的分析方法来描述 peptoids 的序列和结构。串联质谱技术已表明, 作为分析序列控制聚合物序列特性的选择方法, 包括 peptoids11、12、13、 14,15。然而, 系统研究相关的 peptoid 离子碎片模式产生的质谱研究和结构信息的 peptoids 是非常有限的。
Peptoids 可以很容易地合成使用固相方法。成熟的方法包括两步单体加法循环的迭代16,17。在每个加法周期中, 树脂束缚胺是由卤乙酸酸 (通常溴乙酸酸, 乙酰), 这是其次是与主胺的位移反应。尽管自动合成协议已被例行地应用于 peptoid 合成, 但在标准化学实验室中, peptoids 可以人工合成, 并具有优异的产量16,18,19,20。
本实验室采用人工 peptoid 合成方法, 简化了现有方法中使用的仪器。我们以前研究了一系列 peptoids 的碎片模式, 使用 ms/ms 技术21,22,23。我们的结果表明, 当它们受到碰撞诱导的离解 (CID)21,23或电子捕获离解 (peptoids)22实验时, 产生特征碎裂。在本文中, 我们展示了如何在标准化学实验室中合成寡 peptoids, 如何使用三重四极质谱仪进行 CID 实验, 以及如何分析光谱数据。合成的 peptoid 和特征是 nonamer 与 n-末端乙酰化和 C 末端酰, Ac Nae-(北角 Nme)4-NH2。peptoid 的结构显示在图 1中。

<table><tbody><tr><td>ESI-triple quadrupole mass spectrometer, Varian 320L</td><td>Agilent Technologies Inc.</td><td></td><td>The mass spectrometer was acquired from Varian, Inc.</td></tr><tr><td>Varian MS workstation, Version 6.9.2, a data acquisition and data review software</td><td>Varian Inc.</td><td></td><td>The software is a part of the Varian 320L package</td></tr><tr><td>Burrell Scientific Wrist-action shaker, Model 75 DD</td><td>Fisher Scientific International Inc.</td><td>14-400-126</td><td></td></tr><tr><td>Hermle Centrifuge, Model Z 206 A</td><td>Hermle Labortechnik GmbH</td><td></td><td></td></tr><tr><td>Solid phase reaction vessel, 10 mL</td><td>Torviq</td><td>SF-1000</td><td></td></tr><tr><td>Pressure caps for reaction vessels</td><td>Torviq</td><td>PC-SF</td><td></td></tr><tr><td>Syringe filters, pore size 0.2 &amp;mu;m</td><td>Fisher Scientific Inc.</td><td>03-391-3B</td><td></td></tr><tr><td>Syringe filters, pore size 0.45 &amp;mu;m</td><td>Fisher Scientific Inc.</td><td>03-391-3A</td><td></td></tr><tr><td>Polypropylene centrifuge tuges, 50 mL</td><td>VWR International, LLC.</td><td>490001-626</td><td></td></tr><tr><td>Polypropylene centrifuge tuges, 15 mL</td><td>VWR International, LLC.</td><td>490001-620</td><td></td></tr><tr><td>ChemBioDraw, Ultra, Version 12.0</td><td>CambridgeSoft Corporation</td><td></td><td>CambridgeSoft is now part of PerkinElmer Inc.</td></tr><tr><td>Styrofoam cup, 12 Oz</td><td>Common Supermarket</td><td></td><td></td></tr><tr><td>Rink amide resin</td><td>Chem-Impex International, Inc.</td><td>10619</td><td></td></tr><tr><td>Piperidine</td><td>Chem-Impex International, Inc.</td><td>02351</td><td>Highly toxic</td></tr><tr><td>N, N&amp;rsquo;-diisopropylcarbodiimide</td><td>Chem-Impex International, Inc.</td><td>00110</td><td>Highly toxic</td></tr><tr><td>Bromoacetic acid</td><td>Chem-Impex International, Inc.</td><td>26843</td><td>Highly toxic</td></tr><tr><td>2-Phenylethylamine</td><td>VWR International, LLC.</td><td>EM8.07334.0250</td><td></td></tr><tr><td>2-Methyoxyethylamine</td><td>Sigma-Aldrich Co. LLC.</td><td>241067</td><td></td></tr><tr><td>N-Boc-ethylenediamine</td><td>VWR International, LLC.</td><td>AAAL19947-06</td><td></td></tr><tr><td>Acetic anhydride</td><td>Sigma-Aldrich Co. LLC.</td><td>252845</td><td></td></tr><tr><td>N, N-dimethylformamide</td><td>VWR International, LLC.</td><td>BDH1117-4LG</td><td>Further distillation before use</td></tr><tr><td>N, N-diisopropylethylamine</td><td>Chem-Impex International, Inc.</td><td>00141</td><td></td></tr><tr><td>Triisopropylsilane</td><td>Chem-Impex International, Inc.</td><td>01966</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Chem-Impex International, Inc.</td><td>00289</td><td>Highly toxic</td></tr><tr><td>Millipore MILLI-Q Academic Water Purification System</td><td>Millipore Corporation</td><td>ZMQP60001</td><td>For generating HPLC grade water</td></tr><tr><td>HPLC-grade Water</td><td></td><td></td><td>Produced from Millipore MILLI-Q&amp;reg; Academic Water Purification System</td></tr><tr><td>Methanol</td><td>Pharmco-Aaper</td><td>339USP/NF</td><td>HPLC grade</td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific International, Inc.</td><td>A998-4</td><td>HPLC grade</td></tr><tr><td>Diethyl ether</td><td>VWR International, LLC.</td><td>BDH1121-19L</td><td>Further distillation before use</td></tr><tr><td>Dichloromethane</td><td>VWR International, LLC.</td><td>BDH1113-19L</td><td>Further distillation before use</td></tr><tr><td>Nitrogen gas</td><td>Fresno Oxygen/Barnes Supply</td><td>NIT 50-C-F</td><td>Ultra high purity, 99.9995%</td></tr><tr><td>Argon gas</td><td>Fresno Oxygen/Barnes Supply</td><td>ARG 50-C-F</td><td>Ultra high purity, 99.9995%</td></tr></tbody></table>

synthesis and mass spectrometry analysis of oligo-peptoids

Peptoids 是由 n-烷基化甘氨酸单元组成的序列控制的多肽模仿寡聚物。在许多潜在的应用中, peptoids 被认为是一种分子信息存储类型。质谱分析已被认为是测序 peptoids 的选择方法。Peptoids 可以通过固体相化学合成, 使用重复的两步反应循环。本文提出了一种人工合成寡 peptoids 的方法, 并用串联质谱 (ms) 技术对 peptoids 序列进行了分析。样品 peptoid 是一个 nonamer 由交替 n-(2-methyloxyethyl) 甘氨酸 (Nme) 和 n-(2-苯) 甘氨酸 (北角), 以及 n-(2-氨基乙基) 甘氨酸 (Nae) 在 n 终点。peptoid 的序列公式为 ac Nae-(Nme)4-NH2, 其中 Ac 为乙酰基组。综合发生在一个商业上可利用的固相反应容器。溜冰场酰胺树脂作为固体支持, 以产生 peptoid 与酰胺组在 C 总站。所产生的 peptoid 产品是使用三极质谱仪耦合到电喷雾电离源进行序列分析。ms 和 ms 的测量产生了一系列的片段离子产生的分离的带电 peptoid。片段离子根据其质量-电荷比 (m-/z) 的值进行排序。根据 peptoid 碎片的方案, 将片段离子的 m-/z 值与理论上预测的片段离子的标称质量进行比较。分析生成带电 peptoid 的碎片模式。分裂模式与中性 peptoid 单体序列相关。在这方面, MS 分析宣读了 peptoids 的序列信息。

图 1中显示了一个 9 peptoid 的结构, 具有 n-端乙酰化、交 Nae (Nme)4-NH2。peptoid 是通过固相法在烧结聚丙烯反应容器中人工合成的。溜冰场酰胺树脂 (0.047 毫摩尔, 84 毫克与装载0.56 毫摩尔/克) 被用来作为坚实的支持, 以产生 peptoid 与酰胺化的 C 总站。peptoid 链是由单体加法的多个循环所建立的。每个单体加法循环包括两个反应步骤, bromoacety...

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Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

寡 peptoids 的合成及质谱分析

本文介绍了用质谱法进行序列分析的 peptoids 的人工合成的一种协议。

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

synthesis-and-mass-spectrometry-analysis-of-oligo-peptoids

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10.6K 次观看.  University of the Pacific. 本文介绍了用质谱法进行序列分析的 peptoids 的人工合成的一种协议。

视频: Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

质谱途径研究蛋白质结构和相互作用的冻干粉

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

科研

化学

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 characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

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 ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

生物化学

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

不同二硫 Connectivities µ-芋螺毒素 PIIIA 异构体的合成与结构测定

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

肽聚糖的液相色谱质谱和生物信息学半定量分析

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

信息承载Peptoids及其序列定向动态共价自组装的合成

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

使用胺还原甲基化进行样品制备和相对定量，用于肽学研究

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the ...

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step toward a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

质量敏感颗粒跟踪，用于表征膜相关的高分子动力学

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

胞外多糖的OLIGO质谱分析（OLIMP）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

生物学

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.