Mass Spectrometry-Based Fast Photochemical Oxidation of Proteins (FPOP) for Higher Order Structure Characterization.
Mass Spectrometry-Based Fast Photochemical Oxidation of Proteins (FPOP) for Higher Order Structure Characterization.
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DOI：
10.1021/acs.accounts.7b00593
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发表时间：
2018-03-20
影响因子：
18.3
通讯作者：
Gross ML
中科院分区：
文献类型：
--
作者：
Li KS;Shi L;Gross ML
蛋白质结构和相互作用的评估对于理解蛋白质结构/功能关系至关重要。与 X 射线晶体学、核磁共振 (NMR) 和冷冻电镜等高分辨率结构工具以及圆二色性、紫外-可见光和荧光光谱等传统低分辨率方法相比，基于质谱 (MS) 的蛋白质足迹通过利用专注于一级结构的蛋白质组学方法提供中高分辨率（即区域和残基特异性见解）。该方法依赖于用化学标签“绘制”反应性和溶剂暴露的氨基酸残基，并使用修饰模式作为自下而上的基于 MS 的蛋白质组学分析的足迹，以推断蛋白质的高级结构。结果可以参考溶液中甚至细胞中的蛋白质，并且与 X 射线晶体学和 NMR 的结果互补。它特别适用于绘制蛋白质-配体界面以及配体结合、突变和聚集引起的构象变化。蛋白质快速光化学氧化 (FPOP) 最初的概念是一种基于羟基自由基的蛋白质足迹，利用脉冲 KrF 激光 (248 nm) 触发过氧化氢水解，产生溶液羟基自由基，随后对蛋白质进行原位修饰。该平台正在扩展以采用包括卡宾在内的其他活性物质。探针的反应性取决于自由基与残基侧链的固有反应性以及残基的溶剂可及性作为三级/四级结构的函数。通过引入适当的清除剂来与羟基自由基自猝灭竞争，初级自由基的寿命显着缩短至~μs。因此，FPOP的采样时间尺度比氢-氘交换和其他依赖非自由基反应的共价标记方法快得多。 FPOP 的短足迹时间尺度为蛋白质结构阐明提供了两个主要优势：1）它允许以最小的结构扰动在其天然或接近天然状态下研究蛋白质； 2）它对蛋白质高级结构的改变表现出高度的敏感性，因为它的采样时间相对于蛋白质构象变化和动态运动来说很短。此外，羟基自由基的共价和不可逆氧化为下游蛋白质组学工作流程和 MS 分析提供了更大的灵活性，允许具有残基特异性信息的高空间分辨率。自 Hambly 和 Gross 于 2005 年发明以来，FPOP 已从概念验证发展成为一种用于研究蛋白质结构的有价值的生物物理工具。在本文中，我们总结了 FPOP 实现快速标记的原理和实验设计，并描述了该技术在蛋白质高阶结构阐明中当前和独特的功能。应用示例包括β淀粉样蛋白自组装的表征、蛋白质-配体相互作用，特别强调蛋白质治疗剂（例如抗体、Fab和adnectin）的表位作图、详细描述残基特异性折叠动力学的蛋白质折叠以及蛋白质灵活性/动力学。此外，随着我们不断开发新型试剂（例如硫酸根阴离子、卡宾二自由基和三氟甲基自由基），基于 FPOP 的氧化足迹的实用性应该会增长。这些反应试剂与当前的 FPOP 平台兼容，并对各种类型的氨基酸残基提供不同的反应性和选择性，为可溶性蛋白以及最终膜结合蛋白的蛋白质高阶结构提供补充见解。
Assessment of protein structure and interaction is crucial for understanding protein structure/function relationships. Compared to high-resolution structural tools including X-ray crystallography, nuclear magnetic resonance (NMR) and cryo-EM, and traditional low-resolution methods such as circular dichroism, UV-Vis and florescence spectroscopy, mass spectrometry (MS)-based protein footprinting affords medium-to-high resolution (i.e., regional and residue-specific insights) by taking advantage of proteomics methods focused on the primary structure. The methodology relies on “painting” the reactive and solvent-exposed amino acid residues with chemical tags and using the pattern of modifications as footprints from analysis by bottom-up MS-based proteomics to deduce protein higher order structures. The outcome can refer to proteins in solution or even in cells and is complementary to those of X-ray crystallography and NMR. It particularly useful in mapping protein-ligand interfaces and conformational changes resulted from ligand binding, mutation and aggregation. Fast photochemical oxidation of proteins (FPOP), in its original conception, is a type of hydroxyl-radical-based protein footprinting that utilizes a pulsed KrF laser (248 nm) to trigger hydrolysis of hydrogen peroxide to produce solution hydroxyl radicals, which subsequently modify the protein in-situ. The platform is expanding to adopt other reactive species including carbenes. The reactivity of the probe depends on the intrinsic reactivity of the radical with the residue side chain and the solvent accessibility of the residue as a function of the tertiary/quaternary structures. By introducing an appropriate scavenger to compete with hydroxyl radical self-quenching, the lifetime of the primary radicals is remarkably shortened to ∼ μs. Thus, the sampling timescale of FPOP is much faster than hydrogen-deuterium exchange and other covalent labeling methods relying on non-radical reactions. The short footprinting timescale of FPOP offers two major advantages for protein structure elucidation:1) it allows the protein to be interrogated in its native or near-native state with minimum structural perturbation; 2) it exhibits high sensitivity toward alterations in protein higher order structures because its sampling time is short with respect to protein conformational changes and dynamic motion. In addition, the covalent and irreversible oxidation by the hydroxyl radical provides more flexibility in the downstream proteomics workflow and MS analysis, permitting high spatial resolution with residue-specific information. Since its invention in 2005 by Hambly and Gross, FPOP has developed from proof-of-concept to a valuable biophysical tool for interrogating protein structure. In this account, we summarize the principles and experimental design of FPOP that enable its fast labeling, and describe the current and unique capabilities of the technique in protein higher order structure elucidation. Application examples include characterization of amyloid beta self-assembly, protein-ligand interactions with a special emphasis on epitope mapping for protein therapeutics (e.g., antibody, Fab and adnectin), protein folding detailed to residue-specific folding kinetics, and protein flexibility/dynamics. Additionally, the utility of FPOP-based oxidative footprinting should grow with our continuing developments of novel reagents (e.g., sulfate radical anion, carbene diradical and trifluoromethyl radical). These reactive reagents are compatible with the current FPOP platform and offer different reactivity and selectivity towards various types of amino acid residues, providing complementary insights into protein higher order structures for soluble proteins and ultimately for membrane-bound proteins.
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DOI：
10.1007/s13361-012-0403-1
发表时间：
2012-08
影响因子：
3.2
作者：
Chen, Jiawei;Cui, Weidong;Giblin, Daryl;Gross, Michael L.
通讯作者：
Gross, Michael L.
DOI：
10.1007/s13361-014-1055-0
发表时间：
2015-03
影响因子：
3.2
作者：
Zhang Y;Rempel DL;Zhang H;Gross ML
通讯作者：
Gross ML
DOI：
10.1002/anie.201706697
发表时间：
2017-11-06
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Cheng M;Zhang B;Cui W;Gross ML
通讯作者：
Gross ML
氢/氘交换MS对蛋白质构象和动力学的分析。
影响因子：
7.4
作者：
Engen, John R.
通讯作者：
Engen, John R.
影响因子：
7.4
作者：
Gau, Brian C.;Chen, Hao;Zhang, Yun;Gross, Michael L.
通讯作者：
Gross, Michael L.