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><tbody><tr><td>Mercury(II) chloride</td><td>Sigma-Aldrich</td><td>429724</td><td>Highly toxic</td></tr><tr><td>Ammonium formate</td><td>Sigma-Aldrich</td><td>516961</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>F0507</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>Fisher</td><td>A512-P500</td><td></td></tr><tr><td>HPLC water</td><td>Fisher</td><td>W5-4</td><td></td></tr><tr><td>HPLC Acetonitrile</td><td>Fisher</td><td>BP2405-1</td><td></td></tr><tr><td>HPLC Methanol</td><td>Fisher</td><td>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

Determination of the Gas-phase Acidities of Oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

寡肽测定气相酸性

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

科研

化学

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

生产二硫键稳定的跨膜肽复合物的结构研究

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

紧凑型量子点的单分子成像

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

工程学

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

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

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

使用氢交换质谱分析蛋白质动力学

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

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

生物化学

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 ...

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics&#8722;even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

氢-铀交换质谱仪（HDX-MS）平台，用于研究肽生物合成酶

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...

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

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

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

用于测定金属结合寡聚肽的金属电子识别和机制的Ion流动性-质谱技术

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

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-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

基于毛细管电泳的氢/氘交换，用于自上而下的质谱法对蛋白质进行构象表征

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

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

寡肽测定气相酸性

生产二硫键稳定的跨膜肽复合物的结构研究

紧凑型量子点的单分子成像

使用氢交换质谱分析蛋白质动力学

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

质谱途径研究蛋白质结构和相互作用的冻干粉

氢-铀交换质谱仪（HDX-MS）平台，用于研究肽生物合成酶

用于测定金属结合寡聚肽的金属电子识别和机制的Ion流动性-质谱技术

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

基于毛细管电泳的氢/氘交换，用于自上而下的质谱法对蛋白质进行构象表征

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

寡肽测定气相酸性

生产二硫键稳定的跨膜肽复合物的结构研究

紧凑型量子点的单分子成像

使用氢交换质谱分析蛋白质动力学

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

质谱途径研究蛋白质结构和相互作用的冻干粉

氢-铀交换质谱仪（HDX-MS）平台，用于研究肽生物合成酶

用于测定金属结合寡聚肽的金属电子识别和机制的Ion流动性-质谱技术

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

基于毛细管电泳的氢/氘交换，用于自上而下的质谱法对蛋白质进行构象表征

汞（II）与Dicysteinyl四肽的络合的电喷雾质谱研究