<p class="jove_content"工作得到了美国国家科学基金会（CHE-0749737）。太平洋大学所提供的的质谱设施，仪器的使用。</p

1。样品溶液的制备<ol><li>首先准备的肽储备液，使用在体积比为1:1的甲醇和水的混合溶剂的6个参考羧酸。原液的浓度为约10 -3 M。 </li><li>称取1毫克的固体肽样品，A 3的CH，在1.5毫升的Eppendorf管中，并加入1.0毫升的甲醇和水的混合溶剂中，并混合使用涡流。 </li><li>称取1毫克二氟乙酸（DFAH）的，并加入1.0毫升的甲醇和水的混合溶剂中，并混合使用涡流。 </li><li>使用相同的步骤，使股票的解决方案等五大参考酸氯乙酸（MCAH），溴乙酸（巴莫特），二氯乙酸（DCAH），二溴乙酸（DBAH），三氟乙酸（TFAH）。 </li><li>绘制肽原液约50微升至1.5 ml Eppendorf管中，并绘制成一个约50微升原液DFAH我Eppendorf管。稀的混合溶液与900微升甲醇和水的混合溶剂中，以达到终浓度为10 -4 M左右此稀释液将被用于质谱法测量作为样品溶液。参考羧酸以及样品溶液的最终浓度的肽的实际比率将被调整的基础上的离子质谱仪中观察到的信号丰度。 </li><li>使用相同的步骤来准备样品溶液中的肽与其他五个参考酸。 </li></ol> 2。质谱测量1:质子结合团簇离子形成<ol><li>质谱法测量的第一个步骤是，以产生稳定的质子结合簇离子与基准酸的肽。 </li><li>本设备设置为负离子MS模式与ESI针头电压为-4.5 kV，毛细管电压在约-35 V，和干燥气体的温度保持在150℃。将Q1峰宽和Q3峰宽刻度尺（峰宽仪器参数，可以用来调整分辨率的山峰。"刻度"设置允许显示窄峰的峰的分离度更好）。毛细管电压和干燥气体的温度可以调节，以提高所观察到的离子丰度。 </li><li>约0.5毫升的样品溶液肽DFAH的注入到1毫升Hamilton注射器，将注射器连接到ESI针入口使用PEEK（聚醚醚酮）管。然后将注射器的注射泵上。打开注射泵注入样品溶液以10微升/分钟的流量与进入ESI针。 </li><li>打开ESI喷针电压激活ESI过程。打开在检测器上。应观察质谱显示器配置文件中的模式。如果显示的是在质心模式，切换到情景模式。观看团簇离子质子结合形成由monitori高峰在M / Z 428。可以通过调整微调仪器的簇离子的信号丰度。其中一个重要的参数是毛细管电压。可以手动改变毛细管电压（通常在-20到-50V的范围内）的峰在m / z 428的丰度最大化。 </li></ol> 3。质谱测量2:CID包围实验<ol><li>接下来的步骤是，执行CID包围实验。 </li><li>一旦集群离子丰度达到所需的值（约100毫伏），仪器切换到MS / MS模式。在这种模式下，Q1的功能作为质量过滤器作为质量分析器分离的团簇离子，Q2充当碰撞室，和Q3功能。 </li><li>设置碰撞气体（氩气，在这种情况下），在0.5毫乇的压力，并在17 eV的碰撞能量。应遵守三个峰的质谱显示窗口。在m / z 428的峰值对应的簇离子，[DFA•H•A 3 CS]。在M / Z = 332和m / z 95的两个峰，分别对应（3 CS¯）的去质子化的肽和去质子化的二氟乙酸（DFA¯）。小峰在m / z 298是一个辅助的去质子化的肽片段。 2分钟， 图3a中获取的CID谱。 </li><li>执行类似的CID的实验和获取的CID谱Mbah的溴乙酸（）， 图3b中的肽供试品溶液。 </li><li>执行类似CID实验，并与其他所有参考氨基酸的肽样品溶液获得的CID谱。将由此产生的CID谱性质上类似于图3a和3b，但将是不同的m / z值和相对峰高。 </li></ol> 4。质谱测量3:动力学方法<ol><li>最后一步是获得SRM光谱。 </li><li>频谱显示切换到质心和仪器设置为选择反应监测（SRM）模式。作为孤立的离 ​​子保持M / Z 428的第一个四极杆（Q1），填写在四个群众（大众荷比）要监视的第三个四极杆（Q3）。的四个群众米/ z为 428（簇离子），M / Z 332（肽离子），M / Z 298（片段的肽离子），和m / z 95（的DFA¯离子）。碰撞气体压力保持在0.5毫乇。 </li><li>将碰撞能量设置为11.7电子伏特，5分钟，获得的光谱。 </li><li>改变它的碰撞能量为17.6电子伏特，5分钟，获得的光谱。 </li><li>变更23.4电子伏特，29.3 eV的碰撞能量，5分钟，获得在两个碰撞能量光谱。 </li><li>执行类似测量与所有其他的参考氨基酸肽。 </li></ol> 5。数据分析<ol><li>复制离子强度从人的价值升SRM光谱到Excel工作表。 </li><li> CID产品离子分支比计算，LN（[¯] / [A I¯]），测量所有6个质子结合集群在所有四个碰撞能量。样品的值示于表1。 </li><li>值绘制的 ln（[¯] / [A I¯]）对I - ΔΔ 酸 H 酸 H 平均的值。这会给四个碰撞能量， 图4a中的数据相对应的4个重复。 </li><li>提取的值通过线性回归的四个地块的斜坡和拦截。在这种情况下，斜坡的正面的价值观和截距为负值。给符号"X"的斜坡和符号"Y"型拦截。其结果示于表2。的Y的值乘以-1，用符号Y&#39;表示的正的值（这将允许在y轴上显示玩正值）。请注意，这种转换是可选的，只要使用相应的值，使情节为下一步。 </li><li>绘制Y&#39;的值对X， 图4b的值。线性回归的情节产生了一个1.706的斜率和截距-0.536。的斜率对应- ΔΔ 酸 H 酸 H平均 。是已知的值Δ 酸 H平均为330.5千卡/摩尔，这是由该组选定的参考羧酸。然后，从斜率获得的肽的气相酸度的值:Δ 酸 H（CH 3）= 332.2千卡/摩尔。 </li></ol>

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没有透露。

成功的肽的气相酸度测量在很大程度上依赖于选择合适的参考羧酸。理想的参考氨基酸结构相似的有机物，拥有良好的气相酸度值。参考酸彼此具有相似的结构。这将确保对于集合中的每个参考酸脱质子化作用类似的熵。参考酸的酸度值接近的肽。对于较短的寡肽的酰胺化的C-末端含有半胱氨酸的卤代羧酸是合适的参考羧酸的。 CID实验走向成功的一个关键步骤是高丰度稳定团簇离子与质子结合形?...

的氨基酸残基的酸度是最重要的热化学性能的影响的结构，反应性，展开折叠的蛋白质的过程9,19。个别氨基酸残基往往表现出不同的有效的酸度，这取决于他们的位置的蛋白质。特别是，位于活性位点的残基常常表现出明显扰动酸度。这样的一个例子是驻留在的硫氧还蛋白超家族中的酶20,21的活性部位的半胱氨酸残基。活性位点的半胱氨酸是不寻常的酸性相比那些在折叠蛋白3-5。曾有人建议，螺旋构象的不寻常的酸度可以具有显着的贡献。有酸碱性肽开展的解决方案，尤其是在水溶液中2,6-8广泛的实验研究。结果往往是复杂的溶剂效应7。事实上，大多数蛋白质的活性位点位于附近的内部区域，在溶剂的影响降到最小9,10。 为了理解内在的多肽和蛋白质的酸碱性，重要的是在无溶剂的环境中进行的研究。在这里，我们介绍一种基于质谱法测定气相酸性。这种方法被称为扩展厨师动力学方法。该方法已成功地应用到了广泛的化学系统的各种热化学性质的测定，如气相酸度，质子亲合力的金属离子亲和，电子亲和势和电离能11-15， 22-26。我们已采用此方法来确定一系列的半胱氨酸聚丙氨酸和半胱氨酸聚甘氨酸寡肽17,18,27气相酸度。这些研究表明，肽链的N-末端半胱氨酸上课是明显比相应的C-末端的酸性。硫醇盐阴离子与螺旋宏观偶极相互作用强烈稳定的螺旋构象的影响很可能是由于前者的高酸度。由于非易失性的，热不稳定的肽的性质，动力学法是最实用的方法，可在目前产生合理的准确酸碱热化学量的肽28。 图1中所示的总体方案和动力学的方法与方程。测定的气相酸性肽（AH）开始，形成了一系列的质子结合簇阴离子[A•H•A I]¯（或[A¯•H•A I¯] ¯），质谱仪的离子源中的区域，其中，A¯A I¯的去质子化形式的肽，并参考氨基酸。参考的酸是已知的气相酸性的有机化合物。参考酸应该有彼此相似的（但不一定是类似的肽）的结构。参考酸的结构之间的相似性，可确保它们之间的去质子化的熵的相似性。质子结合集群 阴离子质量选择和碰撞激活，随后解离的碰撞诱导解离（CID）的实验，得到相应的单体阴离子，甲¯A I¯，与速率常数k和k分别， 如图1a所示。如果二次碎裂是可以忽略不计，CID碎片离子丰度比，[¯] / [A I¯]，代表的速率常数，K / K I的比例大致衡量。根据假设，有没有反向的激活两个解离通道分支比的CID产物离子的离子屏障，LN [甲¯] / [A I¯]个，将线性相关的肽的气相酸性（Δ 酸 H）和那些参考羧酸（Δ 酸 H 一）， 如图1b所示。在此公式中，Δ 酸 H avg为平均参考酸的气相酸性，Δ（ΔS）是熵的项（可假定为恒定的，如果参考酸的结构彼此相似的），R是通用气体常数，T 效率是有效的系统的温度。实际温度是一个经验参数，取决于几个实验变量，包括将碰撞能量。 气相酸度的值确定通过构建两套热动力学的重复。第一组为OB禀通过绘制LN（¯] / [A I¯]）对H I - ΔΔ 酸酸 H 平均 ， 如图4a所示。线性回归将产生一组直线的斜坡X = 1 / RT eff和拦截的Y = - [ - ΔΔ 酸 H 酸 H AVG] / RT 副作用 - Δ（ΔS）/ R。第二组重复，得到作图得到的截获（Y）对相应的道的来自第一组（X）， 如图4b所示。线性回归产生一个新行的坡度Δ 酸 H - 酸 H平均 ΔΔ（ΔS）/ R，截距。 Δ 酸 H的值，然后从斜率获得和熵项，Δ（ΔS），是从拦截。 实验是使用三重四极杆质谱仪电喷雾电离（ESI）离子源接口。质谱仪的示意图如图2所示。的CID的实验进行质量选择与第一级四极杆单元的簇阴离子的质子结合，并允许他们接受泄漏到大约0.5毫乇的压力下被保持在碰撞室的氩原子的碰撞。分解产物离子质量分析的第三个四极杆单元。 CID谱记录m / z范围广泛，足以涵盖所有可能的二次片段在一些碰撞能量。的CID的产品的离子强度的测量仪器通过设置在选择反应监测（SRM）模式，在该模式下，扫描集中在选定的产物离子。的CID的实验是在四个不同的碰撞能量进行，对应于中心的质量分别为1.0，1.5，2.0，和2.5 eV的能量（E 厘米 ）。质量中心的能量计算公式:E 厘米（cm）= E 实验室 [M /（M +）]， 其中E 实验室是在实验室帧的碰撞能量，m是氩气的质量，M是的质子结合的簇离子的质量。 在这篇文章中，我们使用了寡肽阿拉3 CysNH 2（3 CH）为模型化合物。的C-末端是酰胺化的，将是酸性位的半胱氨酸残基的硫醇基（SH）。为成功地进行测量的气相酸度的合适的参考酸的选择是至关重要的。理想的参考氨基酸结构相似的（互相）拥有良好的气相酸度值的有机化合物。参考酸的酸度值接近的肽。 ，六肽A 3 CH卤化carboxyliÇ酸的选择作为参考的酸。的的六个参考羧酸MCAH氯乙酸，溴乙酸（Mbah的），二氟乙酸（DFAH），二氯乙酸（DCAH），二溴乙酸（DBAH），三氟乙酸（TFAH）。他们两个，DFAH和Mbah的，将被用来说明协议。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>Mass Spectrometer</td>
			<td>Varian</td>
			<td>1200 L and 320 L</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Chloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>402923</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Bromoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>B56307</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Difluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>142859</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dichloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>D54702</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dibromoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>242357</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T6508</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

determination of the gas-phase acidities of oligopeptides

位于折叠的蛋白质在不同位置的氨基酸残基往往表现出不同程度的酸度。例如，一个位于或靠近螺旋的N-末端的半胱氨酸残基通常是酸性比在或接近C-末端1-6。虽然大量的实验研究已经进行了简明的阶段，尤其是在水溶液中6-8酸碱性肽，其结果往往是复杂的由溶剂效应7。事实上，大多数的蛋白质的活性部位位于附近的内陆地区溶剂效应已降到9,10。为了理解内在的多肽和蛋白质的酸碱性，重要的是在无溶剂的环境中进行研究。 我们提出了一种方法来测量气相中的寡肽的酸度。我们使用一个含有半胱氨酸的寡肽，丙氨酸3 CysNH <sUB&gt; 2（3 CH）为模型化合物。测量是基于完善的扩展厨师动力学方法（ 图1）11-16。实验进行了使用三重四极杆质谱电喷雾电离（ESI）离子源（ 图2）连接。对于每种肽的样品中，选择了几个参考羧酸。参考酸的有机化合物在结构上类似于已知的气相酸度。的肽和一个参考酸的混合物的溶液被引入到质谱仪，气相质子结合形成肽参考羧酸阴离子簇。质量分离的质子结合的集群，并随后分散，通过碰撞诱导解离（CID）的实验。使用的酸度和簇离子解离动力学之间的关系，由此产生的碎片离子的丰度分析。然后±1的肽的气相酸度定义线性回归的热动力学地块17,18。 该方法可以应用到各种分子的系统，包括有机化合物，氨基酸及其衍生物，寡核苷酸，寡肽。通过比较，实验测量与计算出的值不同的构象气相酸度，酸度的构象的影响进行评估。

<ol><li>的CID包围实验提供信息相比，选定的参考氨基酸的肽的相对酸度。两个有代表性的与两个参考酸，DFAH和Mbah的肽（A 3 CH），CID谱示于图3。在图3a中的肽离子的离子丰度（峰的高度）是弱于的DFA¯，在图3b中，肽离子的离子丰度是强的MBA¯的。两光谱表明，该肽的气相酸度是在这两个参考酸的酸度之间的范围内。 </li><li>气相酸度的肽的定量值，确定从定量CID实验。重...

Watch this Scientific Journal Video about Determination of the Gas-phase Acidities of Oligopeptides at JoVE.com

Determination of the Gas-phase Acidities of Oligopeptides

含半胱氨酸的寡肽的气相酸度的测定进行说明。使用三重四极杆质谱仪进行实验。肽的相对酸度均采用碰撞诱导解离实验，，使用扩展厨师动力学方法确定定量酸度。

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 ...

determination-of-the-gas-phase-acidities-of-oligopeptides

Research

JoVE Journal

Chemistry

11.0K 次观看.  University of the Pacific. 含半胱氨酸的寡肽的气相酸度的测定进行说明。使用三重四极杆质谱仪进行实验。肽的相对酸度均采用碰撞诱导解离实验，，使用扩展厨师动力学方法确定定量酸度。

视频: Determination of the Gas-phase Acidities of Oligopeptides

Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.&#160;

快速高通量直链淀粉测定冻干马铃薯块茎样品

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in ...

科研

化学

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

隔离和脂质A的化学表征革兰氏阴性菌

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

生物分子有序结构由短肽的自组装形成

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

从构造到晶体 - 迈向β桶外膜蛋白的结构解析

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Determination of Carbonyl Functional Groups in Bio-oils by Potentiometric Titration: The Faix Method

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls cause an increase in viscosity (often referred to as &#39;aging&#39;) during storage of bio-oils. As such, carbonyl content has previously been used as a method of tracking bio-oil aging and condensation reactions with less variability than viscosity measurements. Additionally, carbonyls are also responsible for coke formation in bio-oil upgrading processes. Given the importance of carbonyls in bio-oils, accurate analytical methods for their quantification are very important for the bio-oil community. Potentiometric titration methods based on carbonyl oximation have long been used for the determination of carbonyl content in pyrolysis bio-oils. Here, we present a modification of the traditional carbonyl oximation procedures that results in less reaction time, smaller sample size, higher precision, and more accurate carbonyl determinations. While traditional carbonyl oximation methods occur at room temperature, the Faix method presented here occurs at an elevated temperature of 80 &#176;C.

Here we present a potentiometric titration technique for accurately quantifying carbonyl compounds in pyrolysis bio-oils.

该Faix方法:生物油羰基官能团通过电位滴定法测定

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls ...

Determination of Thermodynamic Properties of Alkaline Earth-liquid Metal Alloys Using the Electromotive Force Technique

A novel electrochemical cell based on a CaF2 solid-state electrolyte has been developed to measure the electromotive force (emf) of binary alkaline earth-liquid metal alloys as functions of both composition and temperature in order to acquire thermodynamic data. The cell consists of a chemically stable solid-state CaF2-AF2 electrolyte (where A is the alkaline-earth element such as Ca, Sr, or Ba), with binary A-B alloy (where B is the liquid metal such as Bi or Sb) working electrodes, and a pure A metal reference electrode. Emf data are collected over a temperature range of 723 K to 1,123 K in 25 K increments for multiple alloy compositions per experiment and the results are analyzed to yield activity values, phase transition temperatures, and partial molar entropies/enthalpies for each composition.

This protocol describes the measurement of the electromotive force of alkaline-earth elements in liquid metal alloys at high temperatures (723-1,123 K) to determine their thermodynamic properties, including activity, partial molar entropy, partial molar enthalpy, and phase transition temperatures, over a wide composition range.

用电动势技术测定碱土金属合金的热力学性质

A novel electrochemical cell based on a CaF2 solid-state electrolyte has been developed to measure the electromotive force (emf) of binary alkaline ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

解析高强共聚物芳纶纤维使其力学性能的测定

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

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

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

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

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

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