Name: determination of the gas-phase acidities of oligopeptides
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<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，?...

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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

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