School of Chemistry and Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne , Melbourne, Victoria 3010, Australia.
School of Chemistry, University of New South Wales , Sydney, New South Wales 2052, Australia.
Anal Chem. 2017 Jun 6;89(11):5748-5756. doi: 10.1021/acs.analchem.6b04604. Epub 2017 May 16.
Metabolic bioactivation of many different chemicals results in the formation of highly reactive compounds (chemically reactive metabolites, CRMs) that can lead to toxicity via binding to macromolecular targets (e.g., proteins or DNA). There is a need to develop robust, rapid, and nontargeted analytical techniques to determine the identity of the protein targets of CRMs and their sites of modification. Here, we introduce a nontargeted methodology capable of determining both the identity of a CRM formed from an administered compound as well as the protein targets modified by the reactive metabolite in a single experiment without prior information. Acetaminophen (N-acetyl-p-aminophenol, APAP) and C-APAP were incubated with rat liver microsomes, which are known to bioactivate APAP to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). Global tryptic digestion followed by liquid chromatographic/mass spectrometric (LC/MS) analysis was used to locate "twin" ion peaks of peptides adducted by NAPQI and for shotgun proteomics via tandem mass spectrometry (MS/MS). By the development of blended data analytics software called Xenophile, the identity of the amino acid residue that was adducted can be established, which eliminates the need for specific parametrization of protein database search algorithms. This combination of experimental design and data analysis software allows the identity of a CRM, the protein target, and the amino acid residues that are modified to be rapidly established directly from experimental data. Xenophile is freely available from https://github.com/mgleeming/Xenophile .
许多不同化学物质的代谢生物活化会导致形成高反应性化合物（化学反应代谢物，CRM），这些化合物通过与大分子靶标（如蛋白质或 DNA）结合而导致毒性。因此需要开发强大、快速和非靶向的分析技术来确定 CRM 的蛋白质靶标及其修饰部位。在这里，我们介绍了一种非靶向方法，该方法能够在无需事先了解的情况下，在单个实验中确定从给药化合物形成的 CRM 的身份以及由反应性代谢物修饰的蛋白质靶标。醋氨酚（N-乙酰对氨基酚，APAP）和 C-APAP 与大鼠肝微粒体孵育，已知这些微粒体可将 APAP 生物转化为反应性代谢物 N-乙酰对苯醌亚胺（NAPQI）。进行全局胰蛋白酶消化，然后进行液相色谱/质谱（LC/MS）分析，以定位由 NAPQI 加合的肽的“孪生”离子峰，并通过串联质谱（MS/MS）进行鸟枪法蛋白质组学分析。通过开发名为 Xenophile 的混合数据分析软件，可以确定加合的氨基酸残基的身份，从而消除了对蛋白质数据库搜索算法进行特定参数化的需要。这种实验设计和数据分析软件的组合允许从实验数据中直接快速确定 CRM 的身份、蛋白质靶标以及被修饰的氨基酸残基。Xenophile 可从 https://github.com/mgleeming/Xenophile 免费获得。
