An Integral Activity-Based Protein Profiling Method for Higher Throughput Determination of Protein Target Sensitivity to Small Molecules

Activity-based protein profiling (ABPP) is a chemoproteomic technique that uses small molecule probes to label active enzymes selectively and covalently in complex proteomes. Competitive ABPP, which involves treatment of the active proteome with an analyte of interest, is especially powerful for profiling how small molecules impact specific protein activities. Advances in higher throughput workflows have made it possible to generate extensive competitive ABPP data across diverse biological samples, making this approach highly appealing for characterizing shared and unique proteins affected by perturbations such as drug or chemical exposures. To use the competitive ABPP approach effectively to understand potential adverse effects of chemicals of concern (CoC), a wide range of concentrations may be needed, particularly for chemicals that lack potency or toxicity data. In this work, we present an integral competitive ABPP method that enables target sensitivity determination for different organophosphate (OP) pesticides as model toxicants. Using previously developed OP-ABPs, we optimized conditions for tandem mass tag (TMT) multiplexing of ABPP samples and compared conventional competitive ABPP involving samples at discrete paraoxon concentrations to pooled samples across that same concentration range. We then expanded our approach to compare protein target sensitivities toward two additional OP pesticides, chlorpyrifos oxon and malaoxon. The results showed that differences in integral intensities for the pooled competition sample can be used to evaluate the relative sensitivity of specific proteins without increasing the overall number of samples. For 8 CoC concentrations of interest, this strategy reduced the number of TMT plexes and the corresponding number of LC–MS/MS analyses 3-fold. We envision the integral ABPP (IABPP) method will provide a means to screen diverse chemicals more rapidly to identify both high and low sensitivity protein targets.基于活性的蛋白质谱分析（Activity-based protein profiling, ABPP）是一类化学蛋白质组学技术，可利用小分子探针在复杂蛋白质组中实现活性酶的选择性共价标记。竞争性ABPP（Competitive ABPP）通过将目标分析物作用于活性蛋白质组，可高效解析小分子对特定蛋白质活性的影响，是极具应用价值的技术手段。高通量工作流程的技术进步使得在多样化生物样本中获取大规模竞争性ABPP数据成为可能，这让该方法在表征受药物、化学暴露等扰动影响的共有与独有蛋白质方面极具吸引力。为有效利用竞争性ABPP方法解析关注化学品（Chemicals of Concern, CoC）的潜在不良效应，通常需要设置宽泛的浓度梯度，尤其针对那些缺乏活性或毒性数据的化学品。本研究提出一种整合型竞争性ABPP方法，可针对作为模式毒物的不同有机磷酸酯（Organophosphate, OP）类农药开展靶点敏感性测定。基于已开发的OP-ABP探针，本研究优化了ABPP样本的串联质量标签（Tandem Mass Tag, TMT）多重标记条件，并将传统的离散对氧磷浓度竞争性ABPP方法，与相同浓度范围的混合样本策略进行了对比。随后，本研究将该方法拓展至另外两种OP类农药——毒死蜱氧（chlorpyrifos oxon）和马拉氧磷（malaoxon）的靶点敏感性比较。研究结果表明，无需增加总样本量，仅通过混合竞争样本的整合强度差异，即可评估特定蛋白质的相对敏感性。针对8种目标关注化学品浓度，该策略将TMT多重标记组数及对应的液相色谱-串联质谱（LC-MS/MS）分析量缩减至原有水平的1/3。本研究展望，整合型ABPP（Integral ABPP, IABPP）方法将为快速筛选多样化化学品、精准识别高与低敏感性蛋白质靶点提供有效途径。