Proteoform-Specific Protein Binding of Small Molecules in Complex Matrices.
Proteoform-Specific Protein Binding of Small Molecules in Complex Matrices.
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DOI：
10.1021/acschembio.6b01018
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发表时间：
2017-02-17
影响因子：
4
通讯作者：
Wang D
中科院分区：
文献类型：
--
作者：
Gil G;Mao P;Avula B;Ali Z;Chittiboyina AG;Khan IA;Walker LA;Wang D
表征蛋白质靶标和小分子之间的特定结合对于药物发现至关重要。传统的分析方法需要通过多步层析分离纯化复杂基质中的小分子，这可能会改变它们原来的生物活性。大多数蛋白质经过翻译后修饰，只有某些蛋白质形式具有正确的构象，具有可访问的结构域和可用于小分子结合的残基。我们开发了一种自上而下的以质谱学(MS)为中心的工作流程，用于一步反应后快速评估植物提取物粗品的生物活性。我们的实验区分了共价结合和非共价结合，并定位了目标蛋白的生物活性成分和特定蛋白形式之间的共价结合残基。我们扩展了我们的方法，用纳米流液相色谱选择反应监测(SRM)-MS方法同时鉴定和无标记多重定量粗制植物提取物中的小分子。我们的方法被验证为人血清白蛋白的各种蛋白形式，这在体内小分子的药代动力学中起着关键作用。我们证明了我们的蛋白质形式特异性分析方法在评估植物粗提物中的胸腺醌、研究其在人体血液中的药代动力学以及在组织培养中解释其对人乳腺癌细胞的毒性方面的实用性。
Characterizing the specific binding between protein targets and small molecules is critically important for drug discovery. Conventional assays require isolation and purification of small molecules from complex matrices through multi-step chromatographic fractionation which may alter their original bioactivity. Most proteins undergo posttranslational modification and only certain proteoforms have the right conformation with accessible domains and available residues for small molecule binding. We developed a top-down mass spectrometry (MS) centric workflow for rapid evaluation of the bioactivity of crude botanical extracts after a one-step reaction. Our assay distinguished covalent from non-covalent binding, and mapped the residue for covalent binding between bioactive constituents and specific proteoforms of the target protein. We augmented our approach with a nanoflow liquid chromatography-selected reaction monitoring (SRM)-MS assay for simultaneous identification and label-free multiplex quantitation of small molecules in the crude botanical extracts. Our assay was validated for various proteoforms of human serum albumin, which plays a key role in pharmacokinetics of small molecules in vivo. We demonstrated the utility of our proteoform-specific assay for evaluating thymoquinone in crude botanical extracts, studying its pharmacokinetics in human blood, and interpreting its toxicity to human breast cancer cells in tissue culture.