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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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?...

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="?...

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

Research

JoVE Journal

Chemistry

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 ...

科研

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

摘要

探索更多视频

此视频中的章节

环境质谱可视化纹影摄影的使用

挥发性有机化合物与大气压化学电离四极杆质谱联用

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

寡 peptoids 的合成及质谱分析

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

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

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

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

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

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

汞（II）与Dicysteinyl四肽的络合的电喷雾质​​谱研究

摘要

探索更多视频

此视频中的章节

环境质谱可视化纹影摄影的使用

挥发性有机化合物与大气压化学电离四极杆质谱联用

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

寡 peptoids 的合成及质谱分析

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

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

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

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

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

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

汞（II）与Dicysteinyl四肽的络合的电喷雾质谱研究