Geib Timon, Moghaddam Ghazaleh, Supinski Aimee, Golizeh Makan, Sleno Lekha
Chemistry Department, Université du Québec à Montréal, Montréal, QC, Canada.
Front Chem. 2021 Aug 20;9:736788. doi: 10.3389/fchem.2021.736788. eCollection 2021.
Acetaminophen (APAP) is a mild analgesic and antipyretic used commonly worldwide. Although considered a safe and effective over-the-counter medication, it is also the leading cause of drug-induced acute liver failure. Its hepatotoxicity has been linked to the covalent binding of its reactive metabolite, -acetyl -benzoquinone imine (NAPQI), to proteins. The aim of this study was to identify APAP-protein targets in both rat and mouse liver, and to compare the results from both species, using bottom-up proteomics with data-dependent high resolution mass spectrometry and targeted multiple reaction monitoring (MRM) experiments. Livers from rats and mice, treated with APAP, were homogenized and digested by trypsin. Digests were then fractionated by mixed-mode solid-phase extraction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Targeted LC-MRM assays were optimized based on high-resolution MS/MS data from information-dependent acquisition (IDA) using control liver homogenates treated with a custom alkylating reagent yielding an isomeric modification to APAP on cysteine residues, to build a modified peptide database. A list of putative targets of APAP were screened from data-dependent high-resolution MS/MS analyses of liver digests, previous studies, as well as selected proteins from the target protein database (TPDB), an online resource compiling previous reports of APAP targets. Multiple protein targets in each species were found, while confirming modification sites. Several proteins were modified in both species, including ATP-citrate synthase, betaine-homocysteine -methyltransferase 1, cytochrome P450 2C6/29, mitochondrial glutamine amidotransferase-like protein/ES1 protein homolog, glutamine synthetase, microsomal glutathione -transferase 1, mitochondrial-processing peptidase, methanethiol oxidase, protein/nucleic acid deglycase DJ-1, triosephosphate isomerase and thioredoxin. The targeted method afforded better reproducibility for analysing these low-abundant modified peptides in highly complex samples compared to traditional data-dependent experiments.
对乙酰氨基酚（APAP）是一种在全球广泛使用的温和镇痛药和解热药。尽管它被认为是一种安全有效的非处方药，但它也是药物性急性肝衰竭的主要原因。其肝毒性与活性代谢物N - 乙酰 - 对苯醌亚胺（NAPQI）与蛋白质的共价结合有关。本研究的目的是使用自下而上的蛋白质组学结合数据依赖型高分辨率质谱和靶向多反应监测（MRM）实验，鉴定大鼠和小鼠肝脏中的APAP - 蛋白质靶点，并比较两个物种的结果。用APAP处理过的大鼠和小鼠肝脏匀浆后用胰蛋白酶消化。消化产物在进行液相色谱 - 串联质谱（LC - MS/MS）分析之前，通过混合模式固相萃取进行分级分离。基于使用定制烷基化试剂处理对照肝脏匀浆后从信息依赖型采集（IDA）获得的高分辨率MS/MS数据，优化靶向LC - MRM分析，该试剂在半胱氨酸残基上对APAP产生异构体修饰，以构建修饰肽数据库。从肝脏消化物的数据依赖型高分辨率MS/MS分析、先前的研究以及从靶向蛋白质数据库（TPDB）（一个汇编APAP靶点先前报告的在线资源）中选择的蛋白质中筛选出APAP的假定靶点列表。在确认修饰位点的同时，在每个物种中发现了多个蛋白质靶点。在两个物种中都有几种蛋白质被修饰，包括ATP - 柠檬酸合酶、甜菜碱 - 高半胱氨酸S - 甲基转移酶1、细胞色素P450 2C6/29、线粒体谷氨酰胺酰胺转移酶样蛋白/ES1蛋白同源物、谷氨酰胺合成酶、微粒体谷胱甘肽S - 转移酶1、线粒体加工肽酶、甲硫醇氧化酶、蛋白质/核酸去糖基化酶DJ - 1、磷酸丙糖异构酶和硫氧还蛋白。与传统的数据依赖型实验相比，靶向方法在分析高度复杂样品中的这些低丰度修饰肽时具有更好的重现性。
