MN-S承认来自美国国家科学基金会的支持下，瑞授予CHE 1011859.作者非常感谢三合会质谱设施在北卡罗来纳大学格林斯伯勒的使用的Thermo Fisher Scientific LTQ XL的Orbitrap质谱仪。作者感谢丹尼尔·托德，文森特·西卡，并Brandie Erhmann在北卡罗来纳大学格林斯伯勒分校，了解有关这项工作有益的建议和意见。

注意:使用前请咨询所有相关的材料安全数据表（MSDS）。氯化汞是一种有毒的化学品。个人防护装备（手套，护目镜，以及白大褂）必须交给当它和所有相关的解决方案，可以穿。在处置指定重金属明确标示化学废物瓶的解决方案。 1.制备5mM的脱气甲酸铵缓冲液，pH为7.5 <ol><li>溶解0.1576克的甲酸铵缓冲液450毫升HPLC级水。调整用1M甲酸和1M氢氧化铵7.5上述溶液的pH值。此溶液转移到500ml容量瓶中和HPLC水添加到该校准线作出的5mM甲酸铵溶液。 </li><li>脱气下一个真空系统的5mM甲酸铵缓冲液10分钟，清洗用氩气。重复两次，在氩气中存储的解决方案。上使用的当天，过滤通过0.2微米的过滤器江前缓冲溶液E使用。 </li></ol> 2.准备氯化汞解决方案<ol><li>称取0.2375克汞（Ⅱ）氯化物。溶解在25ml的5mM甲酸铵缓冲，以产生一个中号0.035汞（Ⅱ）氯化物溶液。 </li><li>添加0.214毫升0.035中号汞（Ⅱ）氯化物溶液到毫升5mM的的9.785甲酸铵缓冲以创建一个7.5×10 -4 M溶液。毯用氩气7.5×10 -4 M的汞（II）溶液。 </li></ol> 3.制备葛洲坝股票方案<ol><li>溶解2.0毫克dicysteinyl四肽，CGGC的，在0.118毫升的HPLC级乙腈和然后添加1.0647毫升的5mM甲酸铵，pH值已脱气，在氩，得到5mM的CGGC原液7.5缓冲液中。 </li><li>添加225微升的5mM CGGC原液至1275微升5mM甲酸铵的形成pH为7.5的缓冲，得到7.5×10 -4 M CGGC溶液。 </li></ol> 4.准备的汞（II）和CGGC的各种反应混合物<ol><li>制备的1:0.5的比例的汞（Ⅱ）:CGGC溶液<ol><li>放置255微升的5mM甲酸铵，pH为7.5的缓冲至1.5 ml微量离心管中。加30微升7.5×10 -4 M的汞（Ⅱ）氯化物溶液到1.5 ml离心管与甲酸铵缓冲。 </li><li>涡旋10秒的溶液。然后加入15微升7.5×10 -4 M葛洲坝溶液倒入1.5 ml离心。涡旋10秒的溶液。让该溶液静置10分钟，在注射前进入质谱仪。 </li></ol></li><li> 1比例的汞（Ⅱ）:1- CGGC溶液<ol><li>放置240微升的5mM甲酸铵，pH为7.5的缓冲至1.5 ml微量离心管中。加30微升7.5×10 -4 M的汞（Ⅱ）氯化物溶液到1.5 ml离心管与甲酸铵缓冲。 </li><li>涡旋10秒的溶液。然后加入30微升7.5×10 -4 M葛洲坝解决方案到1.5 ml离心管。如第4.1节所述重复以类似的方式。 </li></ol></li><li> 2比汞（Ⅱ）:1- CGGC溶液<ol><li>放置210微升的5mM甲酸铵，pH为7.5的缓冲至1.5 ml微量离心管中。加30微升7.5×10 -4 M的汞（Ⅱ）氯化物溶液到1.5 ml离心管与甲酸铵缓冲。 </li><li>涡旋10秒的溶液。然后加入60微升7.5×10 -4 M葛洲坝解决方案到1.5 ml离心管。如第4.1节所述重复以类似的方式。 </li></ol></li></ol> 5.制备CEEC原液<ol><li>溶解3.5毫克dicysteinyl四肽，中欧和东欧国家的，在0.145毫升HPLC级乙腈溶解肽。然后添加13.067毫升5mM的甲酸铵，pH值已脱气，在氩7.5缓冲，以产生0.5米中号CEEC的解决方案。 </li><li>涡的解决方案，直到所有肽溶解。添加1.125 ml的0.5的20mM CEEC溶液和0.375 ml的毫5酸铵，pH为7.5的缓冲到1.5ml微量离心管中，得到7.5×10 -5 M CEEC溶液。涡街，直到混合。 </li></ol> 6.制备汞（II）和中欧和东欧国家解的各种反应混合物<ol><li>制备1:0.5的比例汞（二）:中欧和东欧国家的解决方案<ol><li>放置255微升的5mM甲酸铵，pH为7.5的缓冲至1.5 ml微量离心管中。加30微升7.5×10 -4 M的汞（Ⅱ）氯化物溶液到1.5 ml离心管与甲酸铵缓冲。 </li><li>涡旋10秒的溶液。然后加入15微升7.5×10 -4 M CEEC解决方案到1.5 ml离心管。如第4.1节所述重复以类似的方式。 </li></ol></li><li>制备的1:1的比例的汞（Ⅱ）的:CEEC溶液<ol><li>将24081;微升的5mM甲酸铵，pH为7.5的缓冲至1.5 ml微量离心管中。加30微升7.5×10 -4 M的汞（Ⅱ）氯化物溶液到1.5 ml离心管与甲酸铵缓冲。 </li><li>涡旋10秒的溶液。然后加入30微升7.5×10 -4 M CEEC解决方案到1.5 ml离心管。如第4.1节所述重复以类似的方式。 </li></ol></li><li>制备的1:2的比例的汞（Ⅱ）的:CEEC溶液<ol><li>放置210微升的5mM甲酸铵，pH为7.5的缓冲至1.5 ml微量离心管中。加30微升7.5×10 -4 M的汞（Ⅱ）氯化物溶液到1.5 ml离心管与甲酸铵缓冲。 </li><li>涡旋10秒的溶液。然后加入60微升7.5×10 -4 M CEEC解决方案到1.5 ml离心管。如第4.1节所述重复以类似的方式。 </li></ol></li></ol> 7. ANALY由轨道阱ESI质谱查懋汞（II）和CGGC样品的反应混合物<ol><li>准备ESI质谱仪16 <ol><li>绘制100微升的校准标准为500微升玻璃注射器。 </li><li>将注射器在MS泵的注射器托架，连接管，并注入到质谱仪。 </li><li>通过选择文件图标，并输入文件名设置为运行中的文件名。 </li><li>数据采集​​模块中选择获取数据按钮，并收集150扫描。 </li><li>分析色谱图，打开数据处理模块的软件来验证校准标准。打开该模块，进入文件菜单并选择"打开"，然后选择在对话框中的文件。验证在色谱图中的峰关联到质荷的标准比率。 </li><li>通过制定500微升HPLC级甲醇清洁500微升玻璃注射器，然后分配并购乙醇到烧杯中。 </li><li>制定500微升HPLC级甲醇到玻璃注射器和冲洗系统按照步骤7.1.2。 </li><li>选择软件设定参数的方法中设置模块。选择扫描模式菜单，识别分析仪FTMS，然后单击"确定"。然后通过点击实时查看光谱页面上的各种图标，设置以下参数:鞘气流量:10，离子源温度:0，毛细管电压:37 V，管透镜:95 V，喷雾电压:4.20千伏，流速10.00微升/分钟，分析仪:FTMS，扫描次数:150。 </li></ol></li><li>在ESI质谱仪运行葛洲坝样本<ol><li>运行的5mM甲酸铵pH为7.5的缓冲。 <ol><li>将500微升5毫米甲酸铵缓冲到500微升玻璃注射器，把它放入MS泵的注射器摇篮，并连接管。 </li><li>运行缓冲通过管1-2分钟。 </li><li>建立的文件名的运行通过选择文件图标，并输入文件名。 </li><li>在模块中选择获取数据按钮，并收集150扫描。 </li><li>点击运行按钮来停止征收150扫描采集后。 </li><li>打开数据浏览器模块，然后转到文件菜单并选择"打开"，然后选择在对话框中的文件。确认没有峰值在483，683，1163和1363米/ Z都存在类似于肽或汞（II） - 肽复合物。 </li></ol></li><li>运行1:0.5的汞（Ⅱ）:CGGC比溶液。 <ol><li>将250微升1:0.5的汞（Ⅱ）:将试样放入注射器CGGC比率。 </li><li>将注射器插入该MS泵的注射器托架，连接管，并给装置。 </li><li>通过选择文件图标，并输入文件名选择运行一个文件名。 </li><li>按采样数据按钮，数据采集模块，并领取150扫描，然后点击运行按钮停止收集。 </li><li>打开该模块，进入文件菜单并选择"打开"，然后选择在对话框中的文件。验证色谱峰含有包括一个用于仅在葛洲坝肽。 </li><li>通过吸用500μl甲酸铵缓冲液，然后分配的甲酸铵缓冲到烧杯洗涤注射器。 </li><li>选择在MS上的废物按钮和冲洗管道三次，用500μL甲酸铵缓冲。 </li><li>通过吸用500μl甲醇，然后分配甲醇放入烧杯洗涤注射器。 </li><li>冲洗管一次用500μl甲醇。 </li><li>在MS选择负荷检测器按钮。 </li><li>加入500微升甲酸铵缓冲到注射器。 </li><li>将注射器插入该MS泵的注射器托架，连接管，并给装置。 </li><li>选择通过选择文件图标并输入文件名运行缓冲液中的文件名。 </li><li>按在获取数据按钮，并收集150扫描，然后单击停止运行按钮。 </li><li>打开浏览器的数据模块，转到文件菜单并选择"打开"，然后选择在对话框中的文件。验证色谱是无效的，从以前的汞峰:葛洲坝运行。 </li></ol></li><li>运行1:1的汞（Ⅱ）:CGGC比溶液。 <ol><li>将250微升1:1汞（Ⅱ）:CGGC比样品进入注射器。 </li><li>如对于步骤7.2.2.2到7.2.2.15重复类似的方式。 </li></ol></li><li>运行1:2的汞（Ⅱ）:CGGC比溶液<ol><li>将250微升1:2的汞（Ⅱ）:CGGC浓度样品进入注射器。 </li><li>如对于步骤7.2.2.2到7.2.2.15重复类似的方式。 </li></ol></li></ol></li></ol> 8.分析水星的反应混合物和CEEC样品轨道阱ESI质谱<ol><li>在ESI质谱仪运行中东欧国家样本<ol><li>重复分析程序（步骤7.1至7.2），使用CEEC样品和汞（II）和中欧和东欧国家的不同化学计量比的反应混合物。 </li></ol></li></ol>

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There is no disclosure for the work reported.

疏水dicysteinyl四肽CGGC（C 10 H 18 N 4 O 5 S 2; MW = 338）（图1），形成复合物与汞（Ⅱ），如图2和表 1此外，它形成的肽二聚体和三聚体。增量为在反应混合物中的肽的量增加。如由相关联的二聚体[（2M + H）+ = 677]的m / z值和三聚体〔（3M + H）+ = 1015]，CGGC的硫醇基团没有氧化而形成的实验条件下的二硫化物。这些相关CGGC物种的形成可能是由于此四肽的疏水性。 CGGC形成两种类型的复合物与汞对应于1:1的汞（Ⅱ）:肽和1:2的汞（Ⅱ）:(肽）如前面对dicysteinyl三肽7 报告了2络合物。但是，在过量或等效汞（Ⅱ）的存在下，它也FO有效值是2:2 [汞（Ⅱ）] 2:（肽）2络合物。 羧化dicysteinyl四肽中欧和东欧国家（C 16 H 26 N 4 O 9 S 2; MW = 482）（图1）形成复合物与汞（Ⅱ），如图3和表 1对于没有形成中欧和东欧国家二聚体那样容易。该观察的疏水性更强的葛洲坝。媲美CGGC，它形成配合汞对应于1:1的汞（Ⅱ）:肽和1:2的汞（Ⅱ）:(肽）2络合物。然而，与辅助羧酸盐基时，它形成的2:2 [汞（Ⅱ）] 2:（肽）2配合物更容易。此外，在过量的汞时，它形成了2:1 [汞（Ⅱ）] 2:肽复合物和4:2 [汞（Ⅱ）] 4:（肽）2肽复合物，这些都没有为CGGC观察。 所观察到的信号选择为配合f显示摘要ormed为m / z值示于表1。 <img alt="表格1" src="/files/ftp_upload/53536/53536table1.jpg" /> 表水银肽复合信号1. 概述 。在甲酸铵缓冲液，pH值7.5的LTQ /轨道阱质谱色谱水星肽复合物的信号。 我们已经证明，汞（Ⅱ）和两个dicysteinyl四肽的反应形成复合物是依赖于汞（Ⅱ）的初始比率:肽以及辅助结合基团中的四肽dicysteinyl的存在。此外，汞和肽在根据规定的电喷雾条件所形成的复合物的化学计量精确的可以通过使用基于不同的汞同位素分布模式高分辨率ESI质谱测定。 在pept中半胱氨酸反应的IDE与汞（II）中，必须采取预防措施，以防止半胱氨酰硫醇基团的氧化，以形成二硫键。内所描述的协议中，缓冲溶液小心脱气并在氩气下储存。此外，所有的反应样品立即用ESI质谱分析之前制备。 由于在两个四肽，中欧和东欧国家和CGGC之间溶解度的差异，不同浓度的用于制备储备溶液。 CGGC肽的冷冻储备液弄湿乙腈中容易溶解，随后用5 mM的甲酸铵缓冲液，pH 7.5，以产生7.5×10 -4 M CGGC溶液。在中欧和东欧国家的制备在一个较低的浓度，7.5×10 -5 M，之前的汞（Ⅱ）:肽反应混合物的步骤，因为它的溶解度较低的。用于分析汞最佳稀释度（II）配合被认为是因为该肽，并允许溶解度为10 -5 M在质谱仪除去残余物。在合同的葛洲坝集团的解决方案，CEEC残留坚持管，这需要偶尔管更换。 使用ESI质谱法的汞肽复合物的分析的意义在于它软分析物的电离。这有利于分子离子与碎片可忽略不计的分析。如图这项工作中，它可以被用于表征基于所述签名汞同位素分布模式汞肽复合物的化学计量。然而，一个易失性缓冲区系统是必要的用ESI质谱分析。这可能会限制其实际使用用于鉴定需要更少的挥发性溶剂或缓冲介质溶解分析物。 正如我们前面所提到的7,8，ESI质谱法提供了一种精确测定汞和PEPT的化学计量的灵敏的分析工具IDE在指定的电喷雾离子化条件下的水银肽复合物。然而，它需要使用其他方法（例如，1 H，13 C，199汞核磁共振光谱，扩展X射线吸收精细结构，或电位17-18），以提供更精确地确定复合物中的含量的解。 我们已经表明，ESI带有轨道阱质量分析仪可用于分析汞肽复合物。我们预计，这种技术可以朝向其他金属离子及其络合物与各种小化合物的分析应用。这将是用于分析由能够以不同的同位素形式存在的其他金属离子形成配合物特别有用。

Current clinical drugs prescribed for chelation therapy of mercury poisoning1 contain thiol group(s), which is/are responsible for binding and sequestering mercury ions2,3. However, studies have shown that these small thiol compounds [dimercaptosuccinic acid (DMSA) and dimercaptopropane-sulfonic acid (DMPS)] are not optimal for mercury chelation therapy4-6. Therefore, there is a need to understand the association and complex formation tendencies of mercury with thiols to enhance the rational drug design of thiol compounds for mercury chelation. Recently, we reported that n-alkyl and aryl dicysteinyl tripeptides with dithiol groups can serve as effective "double anchors" to accommodate the coordination sites of mercury(II) to form 1:1 mercury(II):peptide and 1:2 mercury(II):(peptide)2 complexes7. Additionally, we studied the effect of increasing cysteinyl residues on complex type formations8. In this study, we investigate the association of mercury(II) with two dicysteinyl tetrapeptides, where the cysteinyl residues are separated by two amino acid residues. In order to evaluate the effect of auxiliary binding groups for mercury, the intervening amino acids are either two glycine (unsubstituted) residues or two glutamic acid (gamma-carboxylated) residues. 
The reaction of cysteinyl peptide with mercury(II) requires conditions that will prevent the oxidation of the cysteinyl thiol groups to form disulfide bonds9. Moreover, the association of mercury(II) with cysteinyl peptides to form various types of mercury-peptide complexes is dependent on the initial ratio of mercury(II): peptide in the reaction mixture7,8. The types of mercury-peptide complexes formed in these reaction mixtures can be analyzed by soft-ionization mass spectroscopy, which is a sensitive analytical tool for determining species interactions between metal ions and peptides10-14. Accordingly, it will provide a profile of the various types of mercuriated peptide adducts that are formed under a specified electrospray ionization condition. Here, we will show how cysteinyl peptides and mercury(II) chloride solutions can be prepared in degassed ammonium formate buffer solution blanketed with argon to minimize oxidation. By reacting varying mole equivalents of mercury(II) with dicysteinyl tetrapeptides, we will show how the initial ratio of mercury(II):peptide has an effect on the types of complexes formed. We will also show how electrospray ionization (ESI) mass spectrometry can be used as a characterization tool to provide an accurate stoichiometry of mercury to peptide in the complexes formed. The associated video protocol will demonstrate the experimental conditions for preparing the mercury complexes, the procedure for analyzing the reaction mixtures under specified electrospray ionization conditions, and the characterization of the stoichiometries of mercury(II)-dicysteinyl tetrapeptide complexes, based on the distinct mercury isotope distribution patterns, by using the ChemCal program15. It is intended to assist those who are interested in using ESI orbitrap mass spectrometry to analyze various complexes formed by metal ions that exist in different isotopic forms.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mercury(II) chloride</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">429724</td>
			<td style="width:167px;">Highly toxic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ammonium formate</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">516961</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formic acid</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F0507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium hydroxide</td>
			<td style="width:71px;">Fisher</td>
			<td>A512-P500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC water</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">W5-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC Acetonitrile</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP2405-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC Methanol</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">A452-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

a study of the complexation of mercury(ii) with dicysteinyl tetrapeptides by electrospray ionization mass spectrometry

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, by electrospray ionization orbitrap mass spectrometry. This strategy is based on previous successful characterization of mercury-dicysteinyl complexes involving tripeptides by utilizing mass spectrometry among other techniques. Mercury(II) chloride and a dicysteinyl tetrapeptide were incubated in a degassed buffered medium at varying stoichiometric ratios. The complexes formed were subsequently analyzed on an electrospray mass spectrometer consisting of a hybrid linear ion- and orbi- trap mass analyzer. The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired in the positive mode and the observed peaks were then analyzed for distinct mercury isotopic distribution patterns and associated monoisotopic peak. This work demonstrates that an accurate stoichiometry of mercury and peptide in the complexes formed under specified electrospray ionization conditions can be determined by using high resolution ESI MS based on distinct mercury isotopic distribution patterns.

<html>进行一项研究，以表征可能汞肽复合物组成了两个四肽，葛洲坝集团和中东欧国家（ 图1）通过ESI质谱法。汞（II）与CGGC或中欧和东欧国家的复合物经汞的混合物反应，调查（Ⅱ）中，在三个不同的摩尔比肽溶液:1:0.5，1:1和1:2（汞（II）的肽） 。汞（Ⅱ）的浓度为7.5×10 -6 M和肽浓度相应变化。 <img alt="图1" src="/files/ftp_upload/53536/53536fig1.jpg" /> 图1. Dicysteinyl肽的结构。在dicysteinyl四肽，葛洲坝集团与中欧和东欧国家的化学结构。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53536/53536fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ENT"FO:保持together.within页="1"&gt; <img alt="图2" src="/files/ftp_upload/53536/53536fig2.jpg" /> 汞的 图2. ESI MS（II）和从含7.5×10 -6 M的 汞离子在甲酸铵缓冲液，含有不同汞离子pH为7.5的溶液CGGC电喷雾电离轨道阱质谱:CGGC化学计量比:（ 甲 ）1:0.5的比例，（B）的1:1的比例，和（C）的1:2的比例。插图显示指定的汞肽复合物的汞同位素模式。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53536/53536fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53536/53536fig3.jpg" /> 图3. 汞ESI MS（II）和东欧国家 。电喷雾电离轨道阱从质谱含7.5×10 -6 M的 汞离子在甲酸铵缓冲液，pH 7.5，含有不同汞离子的溶液:中欧和东欧国家的化学计量比:（A）1:0.5的比例，（B）的1:1的比例， 和 （C）1 :2的比例。插图显示指定的汞肽复合物的汞同位素模式。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53536/53536fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 共收集汞（Ⅱ）配位与CGGC（图2）和中欧和东欧国家（图3）在不同的汞（Ⅱ）的肽化学计量比电喷雾轨道阱质量色谱（1:0.5，1:1和1:2）。所观察到的汞 - 肽复合物的类型显示不同的汞同位素峰（插图），其用于确定在复汞离子的数量以及depro数tonations。例如， 图1b插图显示的汞同位素特征在肽汞加合物，其对应于汞的七个主要的天然存在的同位素:196汞（0.146％），198汞（10.02％），199汞（16.84％） ，汞200（23.13％），201汞（13.22％），202汞（29.80％），204汞（6.85％），并在括号中指出％的天然丰度。两大同位素200 Hg和202汞柱显示2.3的不同相对强度比为1:3。因此这一个汞同位素簇的最强烈的同位素峰构成的单一同位素质量加合物（M / Z = 539）。它相关用两坐标复杂，这是由两个半胱氨酰硫醇的去质子化形成的，以形成[（CGGC-2H +汞柱）+ H] +加合物。此分析制备如下: 对于[（CGGC-2H +汞柱）+ H] + m / z值是当量人到（338 - 2 + 202 + 1）= 539。 图1A插图显示在肽汞加合物，其对应于一个两汞络合物作为计算通过使用ChemCal方案[（2CGGC-4H + 2HG）+ H] +（图4）中的汞同位素特征。理论质子单一同位素质量对应于1077.061一个m / z值，这是在计算出的同位素簇的第九同位素峰。 图1A插图显示对应于1077.1的m / z值，这也是第九峰的同位素峰在观察到的同位素簇。因此，始发加合物对于此同位素簇可以分配给[（2CGGC-4H + 2HG）+ H] +。 <img alt="图4" src="/files/ftp_upload/53536/53536fig4.jpg" /> 图4.理论同位素型态为[（2CGGC-4H + 2HG）+ H] + </SUP&gt;，理论同位素型态为[（2CGGC-4H + 2HG）+ H] +计算作为通过使用ChemCal程序。箭头表示同位素峰。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53536/53536fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53536/53536fig5.jpg" /> 图5.阳离子化加合物，与汞肽复合物相关的一些阳离子的钠和钾加合物。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53536/53536fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图5示出与由CGGC形成的汞肽复合物相关的一些阳离子化钠和钾加合物。 Sodiated加合物22质量单位更大的牛逼韩相应的质子化汞葛洲坝复合物，而钾加成物38质量单位更大。占主导地位的质子CGGC二聚体（M / Z = 677）也形成与钠阳离子物种（M / Z = 699）和氢离子（M / Z = 715）。这进一步证实了形成CGGC二聚体未经半胱氨酰硫醇基的氧化形成二硫化物，这将导致在一个减少2个质量单位的质子化或阳离子化的加合物。 <img alt="图6" src="/files/ftp_upload/53536/53536fig6.jpg" /> 图6.重叠+1和+2价态，与汞肽离子[（CEEC-4H + 2HG）+ H] +在+1和+2充电状态有关叠嶂。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53536/53536fig6large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alT ="图7"SRC ="/文件/ ftp_upload / 53536 / 53536fig7.jpg"/&gt; 图7.理论同位素型态为[（CEEC-4H + 2HG）+ H] +， 理论同位素型态为[（CEEC-4H + 2HG）+ H] +计算作为通过使用ChemCal程序。箭头表示同位素峰。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53536/53536fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图6示出重叠有在1和2的电荷汞中欧和东欧国家加合物有关的峰。它显示了与汞肽离子[（CEEC-4H + 2HG）+ H] +在1电荷和883.这一个m / z值相关联是在用两汞复杂协议作为计算同位素峰[（CEEC-4H + 2HG）+ H]通过使用ChemCal程序（图7）。理论质子单一同位素质量对应于883.032的m / z值。 上述观察到的[（CEEC-4H + 2HG）+ H] +加合物的883.03一个单一同位素峰与含有相应峰表示额外0.5质量单位另一个加合物重叠。与由轨道阱质谱仪取得的极高分辨率，可以假定这些重叠峰对应于加合物带有电荷的2。因此，的单一同位素质量的重叠复杂被电离，可以计算如下 :图8示出了同位素峰之间的M / Z差为0.5，它们之间的质量差是1原子量。因此，充电状态为+2。为了计算汞肽复合物的质量，所述的M / Z的单一同位素峰用的充电状态相乘，然后从两个质子的质量，从而使该配离子正电减去。 计算为+2加合物: 同位素峰之间M / Z差为0.5 同位素峰之间的质量差为1原子量（1中子） Z = 1除以0.5 = 2 M / Z为质子化单同位素峰（883.53×2） - 2 = 1765.06 为质子化单一同位素峰，[（2CEEC-8H + 4HG）+ H] +，上述m / z值与所计算由ChemCal程序作为1765.056（图8）的理论值相一致。 <img alt="图8" src="/files/ftp_upload/53536/53536fig8.jpg" /> 图8。 理论同位素型态为[（2CEEC-8H + 4HG）+ H] +，理论同位素型态为[（2CEEC-8H + 4HG）+ H] +计算作为通过使用ChemCal程序。箭头表示同位素峰。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53536/53536fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 分析汞肽复合物与ESI轨道阱质谱仪的优点是，每一个离子的电荷可以容易地分配，如上所示。含有碱性氨基末端的肽能容易地稳定正电荷。当使用电喷雾离子化和高分辨率质量分析仪，如轨道阱，肽离子与大于1充电的充电状态可确定更容易相比iontrap质量分析仪的分辨率较低。 （图3A和图6）相关联，重叠峰也通过串联MS分析。它并没有显示出任何的MS-MS裂解，这表明所得到的信号属于预期的化合物如上述所讨论的，并且不聚集形成在较高浓度的汞对肽比率工件。

Watch this Scientific Journal Video about A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry at JoVE.com

A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

汞（II）与Dicysteinyl四肽的络合的电喷雾质​​谱研究

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, ...

a-study-complexation-mercury-ii-with-dicysteinyl-tetrapeptides

Nanyang Technological University

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A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

7.8K 次观看.  University of North Carolina at Greensboro. The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

视频: A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

环境质谱可视化纹影摄影的使用

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

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化学

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 ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

基质辅助激光解吸电离飞行时间 (MALDI) 质谱法表征合成聚合物

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

基质辅助激光解吸/电离质谱中增强碳水化合物离子信号的高效样品制备方法

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

聚类诱导解吸/电电质谱法对复杂分子及其在表面的反应分析

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

纳米材料获得的蛋白质和代谢物科罗纳的毛细电电量谱方法

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein&#39;s structure by MS, specific chemical reactions are performed in solution that encode a protein&#39;s structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as &#946;2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.

The experimental procedures for performing diethylpyrocarbonate-based covalent labeling with mass spectrometric detection are described. Diethylpyrocarbonate is simply mixed with the protein or protein complex of interest, leading to the modification of solvent accessible amino acid residues. The modified residues can be identified after proteolytic digestion and liquid chromatography/mass spectrometry analysis.

通过质谱学研究蛋白质高阶结构，与二乙基碳酸酯共价标签

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool ...

Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end approaches to isolate, enrich, and process proteins ahead of MS are critical for successful proteome characterization. Low, inconsistent protein recovery and residual impurities such as surfactants are detrimental to MS analysis. Protein precipitation is often considered unreliable, time-consuming, and technically challenging to perform compared to other sample preparation strategies. These concerns are overcome by employing optimal protein precipitation protocols. For acetone precipitation, the combination of specific salts, temperature control, solvent composition, and precipitation time is critical, while the efficiency of chloroform/methanol/water precipitation depends on proper pipetting and vial manipulation. Alternatively, these precipitation protocols are streamlined and semi-automated within a disposable spin cartridge. The expected outcomes of solvent-based protein precipitation in the conventional format and using a disposable, two-stage filtration and extraction cartridge are illustrated in this work. This includes the detailed characterization of proteomic mixtures by bottom-up LC-MS/MS analysis. The superior performance of SDS-based workflows is also demonstrated relative to non-contaminated protein.

The present protocol describes solvent-based protein precipitation under controlled conditions for robust and rapid recovery and purification of proteome samples prior to mass spectrometry.

有机溶剂基蛋白质沉淀，在质谱分析之前实现强大的蛋白质组纯化

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end ...

汞（II）与Dicysteinyl四肽的络合的电喷雾质​​谱研究

汞（II）与Dicysteinyl四肽的络合的电喷雾质谱研究