Multiplex labeling chemistry methods for protein footprinting

摘要 蛋白质足迹技术是一种功能强大的中分辨率结构生物学技术 评估蛋白质的结构和动力学依赖于“自下而上”的质谱学 检测、鉴定和定量分析产生的小肽(5-15个残基)。 基于蛋白酶的工作流程。在早期，氢-氘交换(HDX)是领先的，后来 羟基足迹(HRF)等不可逆试剂的研究进展 通过辐射分解；或利用过氧化氢的光解方法，以及最近的等离子体，具有 被引进和提炼。因此，PF被常规地应用于理解 蛋白质-配基在溶液中的高级相互作用上的结合，即使是对于像这样的大分子 抗体、大分子复合体和膜蛋白；所有这些都是重要的药物 目标和生物机器。然而，基于HRF的最先进的PF研究通常报告 仅来自上述多肽中20%的可能位点的数据，限制了总体影响。 这在一定程度上是由于含硫(特别是蛋氨酸)和芳香族残留物的高活性 相对于许多其他问题，造成了同时检测高和低的动态范围问题 在同一个实验中有丰富的物种。在这项提案中，响应PAR-19-253，我们 提出一系列新的标记、生物物理学和质谱学方法，基于 以自由基活化的三氟甲基化为基础的化学，提供多重，高密度的 分辨率标记和质谱分析工作流程，以实现价值最大化和 PF研究的影响，读数为50%-100%的可接触侧链 相关多肽。该项目利用了我们先进的同步辐射分解平台和我们的 验证和协作战略将把结果扩展到主要的PF平台，例如： 辐射分解、光解和基于等离子体的HRF。 在目标1(0-20个月)中，我们将对羟基自由基诱导TFM的化学进行基准测试， 通过检测氨基酸了解其与羟基自由基的侧链反应性 以及各种可用的TFM试剂的基于多肽的反应性。这些技术的初步发展 TFM标记方法利用我们先进的HRF同步辐射分解平台，但我们 我将使用辐射分解和光解来验证该方法，并找到最佳条件 在扩大PF覆盖面的同时优化标签的动态范围。里程碑：修改~16/20 一锅反应中的残留物(今天为~12/20)。在单独+16的情况下，动态范围为~100或更小 (-O)和+68(-CF3)通道(目前为+16(-O)通道中的1000通道)。在AIM 2(20个月- ，我们将扩展我们的发现，朗格洛斯试剂是一种很有希望的羟基候选者 自由基诱导的TFM与结构定量探测蛋白质结构 理解并将易处理的钙调蛋白和雌激素受体作为基准目标。 里程碑：优化和验证用于量化结构的简单、易于实施的工作流 基于TFM标记(辐射分解和光解)的结构分辨蛋白质的评价 50%-100%的残基处于可接近残基的多肽水平。论证方法体现了 在目标3(40-60个月)中，我们将探索朗格洛斯和其他 作为足迹驱动小分子结构活性的化学标记方法的试剂 分析，以扩大蛋白质口袋的结构覆盖范围，以优化配体-蛋白质相互作用 分析。里程碑：18/20个残基可能通过多锅反应标记，达到80- 100%覆盖。此外，还将测试和传播技术发展。 通过协作并通过广泛的PF的直接培训，将光束线连接到台式协议 通过出版物和讲习班为社区服务。

Abstract Protein footprinting (PF) is a powerful medium resolution structural biology technique for assessing protein structure and dynamics that relies on “bottom-up” mass spectrometry (MS) to detect, identify, and quantitatively analyze the small (5-15 residue) peptides that are generated in protease-based workflows. Early on hydrogen deuterium exchange (HDX) led the way, later advances in irreversible reagent development, such as hydroxyl radical footprinting (HRF) mediated by radiolysis; or methods utilizing photolysis of peroxide, and recently plasma, have been introduced and refined. As a result, PF is routinely applied in understanding the effects of protein-ligand binding on higher order interactions in solution, even for large macromolecules like antibodies, large molecular complexes, and membrane proteins; all of which are important drug targets and biological machines. However, state of the art HRF-based PF studies typically report data from only <20% of the possible sites within the above peptides, limiting the overall impact. In part this is due to the high reactivity of sulfur containing (Met in particular) and aromatic residues relative to many others, creating dynamic range issues for simultaneously detecting high and low abundant species in the same experiment. In this proposal, responsive to PAR-19-253, we propose a range of novel labeling, biophysics, and mass spectrometry methods, based on radical activated trifluoromethylation based chemistries, to provide multiplex, high- resolution labeling and mass spectrometry analysis workflows to maximize value and impact of PF studies, with readouts from 50-100% of accessible side chains within the relevant peptides. The project leverages our advanced synchrotron radiolysis platform and our validation and collaboration strategies will extend the results to major PF platforms such as: radiolysis, photolysis, and plasma based HRF. In Aim 1 (months 0-20), we will benchmark the chemistry of hydroxyl radical induced TFM, understanding its side chain-based reactivity compared to OH radical by examining amino acid and peptide based reactivity for a variety of available TFM reagents. Initial developments of these TFM labeling approaches leverage our advanced synchrotron radiolysis platform for HRF, but we will validate the method using both radiolysis and photolysis, and finding optimum conditions for expanding PF coverage while optimizing dynamic range of labeling. Milestones: Modify ~16/20 residues in a one-pot reaction (vs ~12/20 today). Dynamic range of ~100 or less in separate +16 (-O) and +68 (-CF3) channels (vs. 1000-today in one +16 (-O) channel). In Aim 2 (months 20- 40), we will extend our findings that the Langlois reagent is a promising candidate for hydroxyl radical induced TFM and probing protein structure in a quantitative way using structurally understood and PF tractable calmodulin and estrogen receptor as benchmarked targets. Milestone: Optimize and validate a simple, easy to implement workflow for quantitative structure assessment of proteins based on TFM labeling (radiolysis and photolysis) at structural resolution of 50-100% of residues at the peptide level of accessible residues. Demonstrate method reflects structure at an accuracy of +/- 30Å2. In Aim 3 (months 40-60) we will explore Langlois and other reagents as chemical labeling approaches for footprinting driven small molecule structure activity analysis, to expand structural coverage of protein pockets for optimizing ligand-protein interaction analysis. Milestone: 18/20 residues potentially labelled through multi-pot reactions achieving 80- 100% coverage. In addition, the technology developments will be tested and disseminated in beamline to benchtop protocols with collaborations and through direct training of a wide PF community through publications and workshops.