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Chimie

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

Published: September 07, 2019
doi:
10.3791/60102
, , , ,
1Department of Chemistry,Texas A&M University-Commerce

Summary

Ion 流动性质谱和分子建模技术可以描述设计金属结合肽和铜结合肽美他可辛的选择性金属结合性能。开发新的金属包合肽类将有助于治疗与金属子失平衡相关的疾病。

Abstract

电喷雾电位化（ESI）可以将水相肽或肽复合物转移到气相，同时保存其质量、总电荷、金属结合相互作用和构象形状。ESI 与电子流动性质谱法 （IM-MS） 耦合提供了一种工具技术，允许同时测量肽的量位 （m/z） 和碰撞横截面 （CCS），和构象形状。肽复合物的总体电荷由肽的酸性和基本位点1）和2）金属电的氧化状态控制。因此，复合物的总体电荷状态是影响肽金属电结合亲和力的溶液的pH的函数。对于 ESI-IM-MS 分析，肽和金属离子溶液由仅水溶液制备，pHH 与稀水醋酸或氢氧化铵一起调整。这允许为特定的肽确定pH依赖性和金属电位选择性。此外，肽复合物的m/z和CCS可与B3LYP/LanL2DZ分子建模一起使用，以识别该复合物的金属电协调结合点位和三级结构。结果表明，ESI-IM-MS如何描述一组替代的美他可辛肽的选择性结合性能，并将其与铜结合肽美他可曲蛋白进行比较。

Introduction

铜和锌离子对生物体至关重要，对包括氧化保护、组织生长、呼吸、胆固醇、葡萄糖代谢和基因组读数1在内的过程至关重要。为了启用这些功能，组，如Cys的硫酸盐，他的2，3，（更罕见）的硫丹，和Glu和Asp的甲苯选择性地将金属作为辅助因子到活动位点金属酶。这些协调小组的相似性提出了一个耐人寻味的问题，即他和Cys配体如何选择性地将Cu（I/II）或Zn（II）纳入，以确保正确的功能。

选择性结合通常通过获取和贩运肽来完成，这种肽控制锌（II）或Cu（I/II）的i/II）子浓度4。Cu（I/II）具有高度反应性，对酶造成氧化损伤或异化结合，因此其自由浓度受到铜伴和铜调节蛋白的严格调节，这些蛋白质安全地将其输送到细胞中的各个位置，并紧密地调节控制其平衡5，6。铜代谢或平衡的中断与门克斯和威尔逊的疾病7以及癌症7和神经紊乱直接相关，如普里昂8和阿尔茨海默氏病9。

Wilson 的疾病与眼睛、肝脏和大脑部分的铜含量增加有关，其中 Cu（I/II） 的氧化还原反应产生活性氧，导致肝神经退化。现有的包合疗法是小的三醇氨基酸阴西胺和三甲四胺。或者，甲抗凝铜采集肽甲酰沙棘素（mb）10，11表现出治疗潜力，因为它们对Cu（I）12具有高结合亲和力。当甲基三磷酸三甲杆菌OB3b的甲胺素（mb-OB3b）在威尔逊病的动物模型中被研究时，铜有效地从肝脏中取出，并通过胆汁13排泄。体外实验证实，mb-OB3b可以从肝细胞醇13中所含的铜金属醇素中洗去铜。激光消融电耦合等离子体质谱成像技术研究了威尔逊病肝样本14、15、16中铜的空间分布，并表明mb-OB3b去除铜与短处理期只有8天17。

mb-OB3b还将与其他金属离子结合，包括Ag（I）、Au（III）、Pb（II）、Mn（II）、Co（II）、Fe（II）、Ni（II）和Zn（II）18、19。Ag（I）展示的生理Cu（I）结合位点的竞争，因为它可以取代Cu（I）从mb-OB3b复合体，与Ag（I）和Ni（II）也表现出不可逆转的绑定Mb，不能取代Cu（I）19。最近，研究了一系列具有2His-2Cys结合图案的甲酰亚布辛（amb）寡肽，其锌（II）和Cu（I/II）结合特性具有特征。他们的主要氨基酸序列是相似的，他们都包含2His-2Cys主题，Pro和乙酰化N-总站。它们主要不同于mb-OB3b，因为2His-2Cys图案取代了mb-OB3b的两个enethiol牛酮结合位点。

电喷雾电位化与电力流动性质谱（ESI-IM-MS）相结合，为确定肽的金属结合特性提供了强大的工具技术，因为它测量其质量电荷（m/z）和碰撞横截面 （CCS），同时从溶液相中保存其质量、电荷和构象形状。m/z和 CCS 与肽体测定学、原形状态和构象形状有关。由于明确确定了物种中每个元素的身份和数量，因此确定骨化学计量。肽复合物的总体电荷与酸性和基本位点的原位和金属电的氧化状态有关。CCS提供了肽复合物的构象形状信息，因为它测量了与复体三级结构相关的旋转平均大小。复合物的整体电荷状态也是pH的函数，影响肽的金属电结合亲和力，因为脱质子的基本或酸性位点，如carboxyl、His、Cys和Tyr也是金属电的的潜在结合位点。在分析中，肽和金属离子在水溶液中制备，pH由稀水性醋酸或氢氧化铵调整。这允许为肽确定pH依赖性和金属电位选择性。此外，ESI-IM-MS测定的m/z和CCS可与B3LYP/LanL2DZ分子建模一起使用，以发现复合体金属电一致的类型和三级结构。本文中显示的结果揭示了ESI-IM-MS如何描述一组amb肽的选择性包合性能，并将其与铜结合肽mb-OB3b进行比较。

Protocol

1. 试剂制备 培养甲基肌酸三角性四氯环带，分离无Cu（I）的mb-OB3b 18，22，23，冷冻干燥样品，并储存在-80°C，直到使用。 合成 amb 肽（>98% 纯度为 amb1，amb2，amb4;> 70% 纯度为 amb7），冷冻干燥样品，并将其储存在 -80°C 直到使用。 购买 >98% 纯度氯化锰（II）氯化物、?…

Representative Results

amb1的金属绑定Amb1的 IM-MS 研究20（图 1A） 显示，铜离子和锌离子以 pH 依赖性的方式结合到 amb1（图 2）。然而，铜和锌通过不同协调部位的不同反应机制与amb1结合。例如，将Cu（II）添加到amb1中，通过二硫化物桥形成导致amb1（amb 1ox）氧化，在pH>6时?…

Discussion

关键步骤：通过 ESI-IM-MS 保存用于检查的解决方案阶段行为
必须使用本机 ESI 仪器设置来保存肽的化学测量、电荷状态和构象结构。对于原生条件，必须优化 ESI 源中的条件，如锥电压、温度和气体流量。此外，必须检查源、陷阱、电子移动性和传输移动波（尤其是控制注入电压进入 IM 单元的直流陷印偏置）中的压力和电压，以检查其对充电状态和电子移动分布的影响。

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Divulgations

The authors have nothing to disclose.

Acknowledgements

此材料基于国家科学基金会支持的工作，包括 1764436、NSF 仪器支持 （MRI-0821247）、韦尔奇基金会 （T-0014） 以及能源部的计算资源 （TX-W-20090427-0004-50） 和 L3 通信.我们感谢鲍尔的加州大学圣巴巴拉分校小组分享了西格玛计划，阿约巴米·伊莱桑米在视频中演示了这项技术。

Materials

acetonitrile HPLC-grade Fisher Scientific (www.Fishersci.com) A998SK-4
ammonium hydroxide (trace metal grade) Fisher Scientific (www.Fishersci.com) A512-P500
cobalt(II) chloride hexahydrate 99.99% Sigma-Aldrich (www.sigmaaldrich.com) 255599-5G
copper(II) chloride 99.999% Sigma-Aldrich (www.sigmaaldrich.com) 203149-10G
copper(II) nitrate hydrate 99.99% Sigma-Aldrich (www.sigmaaldrich.com) 229636-5G
designed amb1,2,3,4,5,6,7 peptides Neo BioLab (neobiolab.com) designed peptides were synthized by order
designed amb5B,C,D,E,F peptides PepmicCo (www.pepmic.com) designed peptides were synthized by order
Driftscope 2.1 software program Waters (www.waters.com) software analysis program
Freeze-dried, purified, Cu(I)-free mb-OB3b cultured and isolated in the lab of Dr. DongWon Choi (Biology Department, Texas A&M-Commerce)
glacial acetic acid (Optima grade) Fisher Scientific (www.Fishersci.com) A465-250
Iron(III) Chloride Anhydrous 98%+ Alfa Aesar (www.alfa.com) 12357-09
lead(II) nitrate ACS grade Avantor (www.avantormaterials.com) 128545-50G
manganese(II) chloride tetrahydrate 99.99% Sigma-Aldrich (www.sigmaaldrich.com) 203734-5G
MassLynx 4.1 Waters (www.waters.com) software analysis program
nickel chloride hexahydrate 99.99% Sigma-Aldrich (www.sigmaaldrich.com) 203866-5G
poly-DL-alanine Sigma-Aldrich (www.sigmaaldrich.com) P9003-25MG
silver nitrate 99.9%+ Alfa Aesar (www.alfa.com) 11414-06
Waters Synapt G1 HDMS Waters (www.waters.com) quadrupole – ion mobility- time-of-flight mass spectrometer
zinc chloride anhydrous Alfa Aesar (www.alfa.com) A16281
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Tags

Keywords: Ion Mobility-mass SpectrometryIM-MSMetal Ion RecognitionRedox ActivityMetal Binding OligopeptidesMolecular ModelingTertiary StructureMetal CorrelationESI-IM-MSPoly-DL-alanineAlternative MethanobactinPH-dependent RedoxMethyl Binding ReactionProduct IonsStoichiometryProtonation StateConformational StructureMetal Chelating PeptidesTherapeuticsMenkes DiseaseWilson’s DiseaseCancerAlzheimer’s DiseaseNative ESI-IM-MSNegative IonPositive IonAcetic AcidAmmonium HydroxideMass-to-chargeIsotope Pattern
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Yousef, E. N., Sesham, R., McCabe, J. W., Vangala, R., Angel, L. A. Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides. J. Vis. Exp. (151), e60102, doi:10.3791/60102 (2019).
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