Multiplex labeling chemistry methods for protein footprinting

摘要 蛋白质足迹（PF）是一种强大的中等分辨率结构生物学技术， 评估蛋白质结构和动力学，依赖于“自下而上”的质谱法（MS）， 检测，鉴定和定量分析小（5-15个残基）肽， 基于蛋白酶的工作流程。早期氢氘交换（HDX）引领了这一方向， 不可逆试剂开发的进展，如羟基自由基足迹法（HRF） 通过辐解介导的方法;或利用过氧化物的光解和最近的等离子体的方法， 被引进和完善。因此，PF通常用于理解 蛋白质-配体结合在溶液中的高阶相互作用上，即使对于大的大分子，如 抗体、大分子复合物和膜蛋白;所有这些都是重要的药物 目标和生物机器然而，基于HRF的PF研究通常报告 仅来自上述肽内<20%的可能位点的数据，限制了总体影响。 这部分是由于含硫（特别是Met）和芳族残基的高反应性 相对于许多其他的，创建动态范围问题，同时检测高和低 在同一个实验中，在本提案中，响应PAR-19-253，我们 提出了一系列新的标记，生物物理学和质谱方法，基于 基于自由基活化的三氟甲基化的化学，以提供多重的，高- 分辨率标记和质谱分析工作流程，以实现价值最大化， PF研究的影响，从50-100%的可接近的侧链内读出 相关肽。该项目利用我们先进的同步加速器辐解平台和我们的 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的TFM的化学进行基准测试， 通过检查氨基酸，了解其与OH自由基相比基于侧链的反应性 和基于肽的反应性。这些方面的初步发展 TFM标记方法利用我们先进的HRF同步辐射分解平台，但我们 将使用辐解和光解来验证该方法，并找到最佳条件， 扩大PF覆盖范围，同时优化标记的动态范围。里程：修改~16/20 在一锅反应中的残留物（与今天的约12/20相比）。动态范围约为100或更小，单独+16 (-O)和+68（-CF 3）通道（与今天的1000个+16（-O）通道相比）。在目标2（20- 40），我们将扩展我们的研究结果，即Langlois试剂是一个有前途的候选人羟基 自由基诱导的TFM和探测蛋白质结构的定量方法， 了解和PF易处理的钙调蛋白和雌激素受体作为基准目标。 里程碑：优化和验证一个简单、易于实施的定量结构工作流程 基于TFM标记（辐解和光解）在结构分辨率下评估蛋白质 50-100%的残基在肽水平的可接近残基。示范法体现 精度为+/-30 μ m 2。在目标3（40-60个月），我们将探讨Langlois和其他 作为足迹法驱动的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种残留物可能通过多锅反应标记，达到80- 100%覆盖。此外，将在2010年测试和传播技术发展， 通过协作和直接训练广泛的PF， 通过出版物和研讨会。

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.