Proteomic characterisation of drug metabolising enzymes and drug transporters in pig liver

Liver enzymes and transporters play an essential role in xenobiotic metabolism, distribution and elimination. Pre-clinical safety assessment relies on studies on animal models, including the (mini)pig. The pig shares many anatomical and physiological characteristics with humans, and there is currently a gap in information about porcine metabolism and disposition pathways and their similarities and differences from human ones.Three different sample preparation methods (filter-aided sample preparation (FASP), enhanced FASP (eFASP) and in-solution sample preparation) were used to prepare porcine liver tissue (two samples) for proteomic analysis. The analysis relied on rapid-separation liquid chromatography coupled to Orbitrap mass spectrometry in data-dependent acquisition mode. MASCOT was used for identification and relative label-free quantification was based on spectral counting.The three sample preparation methods provided complementary results, allowing characterisation of approximately 70 pharmacologically relevant proteins. The main quantified proteins included 16 cytochrome P450 (CYP) enzymes, 5 UGT enzymes, and 11 transporters. In addition, 20 Phase I and 14 Phase II enzymes were also characterised. Inter-operator differences were negligible and the pig liver pies for CYP, UGT and efflux transporter proteins were established. Human homologues of the quantified CYP, UGT and transporter proteins were identified. Liver enzymes and transporters play an essential role in xenobiotic metabolism, distribution and elimination. Pre-clinical safety assessment relies on studies on animal models, including the (mini)pig. The pig shares many anatomical and physiological characteristics with humans, and there is currently a gap in information about porcine metabolism and disposition pathways and their similarities and differences from human ones. Three different sample preparation methods (filter-aided sample preparation (FASP), enhanced FASP (eFASP) and in-solution sample preparation) were used to prepare porcine liver tissue (two samples) for proteomic analysis. The analysis relied on rapid-separation liquid chromatography coupled to Orbitrap mass spectrometry in data-dependent acquisition mode. MASCOT was used for identification and relative label-free quantification was based on spectral counting. The three sample preparation methods provided complementary results, allowing characterisation of approximately 70 pharmacologically relevant proteins. The main quantified proteins included 16 cytochrome P450 (CYP) enzymes, 5 UGT enzymes, and 11 transporters. In addition, 20 Phase I and 14 Phase II enzymes were also characterised. Inter-operator differences were negligible and the pig liver pies for CYP, UGT and efflux transporter proteins were established. Human homologues of the quantified CYP, UGT and transporter proteins were identified.

肝脏酶类与转运蛋白在外源性物质的代谢、分布及清除过程中发挥关键作用。临床前安全性评价依赖于动物模型研究，其中包括（迷你）猪。猪在解剖学与生理学特征上与人类高度相似，但目前关于猪的代谢与处置通路，及其与人类通路的异同点的相关信息仍存在缺口。三种不同的样品制备方法——滤膜辅助样品制备（filter-aided sample preparation, FASP）、增强型FASP（enhanced FASP, eFASP）以及溶液内样品制备——被用于处理两份猪肝脏组织样本，以开展蛋白质组学分析。本分析采用数据依赖性采集模式下的快速分离液相色谱联用Orbitrap轨道阱质谱技术。采用MASCOT软件进行蛋白质鉴定，相对无标记定量则基于光谱计数法实现。三种样品制备方法所得结果互为补充，成功鉴定了约70种药理学相关蛋白质。所定量的核心蛋白质包括16种细胞色素P450（cytochrome P450, CYP）酶、5种尿苷二磷酸葡萄糖醛酸转移酶（UDP-glucuronosyltransferase, UGT）以及11种转运蛋白。此外，本研究还鉴定了20种I相代谢酶与14种II相代谢酶。操作者间的差异可忽略不计，猪肝脏中CYP、UGT以及外排转运蛋白的表达谱得以建立。本研究同时鉴定了所定量的CYP、UGT以及转运蛋白的人类同源蛋白。

肝脏酶类与转运蛋白在外源性物质的代谢、分布及清除过程中发挥关键作用。临床前安全性评价依赖于动物模型研究，其中包括（迷你）猪。猪在解剖学与生理学特征上与人类高度相似，但目前关于猪的代谢与处置通路，及其与人类通路的异同点的相关信息仍存在缺口。三种不同的样品制备方法——滤膜辅助样品制备（filter-aided sample preparation, FASP）、增强型FASP（enhanced FASP, eFASP）以及溶液内样品制备——被用于处理两份猪肝脏组织样本，以开展蛋白质组学分析。本分析采用数据依赖性采集模式下的快速分离液相色谱联用Orbitrap轨道阱质谱技术。采用MASCOT软件进行蛋白质鉴定，相对无标记定量则基于光谱计数法实现。三种样品制备方法所得结果互为补充，成功鉴定了约70种药理学相关蛋白质。所定量的核心蛋白质包括16种细胞色素P450（cytochrome P450, CYP）酶、5种尿苷二磷酸葡萄糖醛酸转移酶（UDP-glucuronosyltransferase, UGT）以及11种转运蛋白。此外，本研究还鉴定了20种I相代谢酶与14种II相代谢酶。操作者间的差异可忽略不计，猪肝脏中CYP、UGT以及外排转运蛋白的表达谱得以建立。本研究同时鉴定了所定量的CYP、UGT以及转运蛋白的人类同源蛋白。