Accurate measurement of drug-target interactions in vivo is critical for both preclinical development and translation to clinical studies, yet many assays rely on indirect measures such as biomarkers associated with target activity. Activity-based protein profiling (ABPP) is a direct method of quantifying enzyme activity using active site-targeted small-molecule covalent probes that selectively label active but not inhibitor-bound enzymes. Probe-labeled enzymes in complex proteomes are separated by polyacrylamide gel electrophoresis and quantified by fluorescence imaging. To accelerate workflows and avoid imaging artifacts that make conventional gels challenging to quantify, we adapted protocols for a commercial LabChip GXII microfluidic instrument to permit electrophoretic separation of probe-labeled proteins in tissue lysates and plasma, and quantification of fluorescence (probe/protein labeling ratio of 1:1). Electrophoretic separation on chips occurred in 40 s per sample, and instrument software automatically identified and quantified peaks, resulting in an overall time savings of 3-5 h per 96-well sample plate. Calculated percent inhibition was not significantly different between the two formats. Chip performance was consistent between chips and sample replicates. Conventional gel imaging was more sensitive but required five times higher sample volume than microfluidic chips. Microfluidic chips produced results comparable to those of gels but with much lower sample consumption, facilitating assay miniaturization for scarce biological samples. The time savings afforded by microfluidic electrophoresis and automatic quantification has allowed us to incorporate microfluidic ABPP early in the drug discovery workflow, enabling routine assessments of tissue distribution and engagement of targets and off-targets in vivo.

中文翻译：

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使用基于微流体活性的蛋白质谱分析对体内靶标进行定量分析。

体内药物-靶标相互作用的准确测量对于临床前开发和临床研究都是至关重要的，但是许多测定方法都依赖于间接测量，例如与靶标活性相关的生物标志物。基于活性的蛋白质谱分析（ABPP）是一种使用活性位点靶向的小分子共价探针定量酶活性的直接方法，该探针选择性标记活性而不是抑制剂结合的酶。复杂蛋白质组中的探针标记酶通过聚丙烯酰胺凝胶电泳分离，并通过荧光成像进行定量。为了加快工作流程并避免使常规凝胶难以定量的成像假象，我们针对商业LabChip GXII微流控仪器调整了方案，以允许电泳分离组织裂解液和血浆中探针标记的蛋白质，和荧光定量（探针/蛋白质标记比例为1：1）。每个样品在芯片上进行电泳分离的时间为40 s，仪器软件自动识别和定量峰，每块96孔样品板总共可节省3-5 h的时间。两种形式之间的计算出的抑制百分比没有显着差异。芯片和样品重复样品之间的芯片性能一致。常规的凝胶成像更灵敏，但需要的样品量是微流控芯片的五倍。微流控芯片产生的结果与凝胶相当，但样品消耗量低得多，从而有助于将稀有生物样品的测定小型化。