矩阵热转移技术鉴定有机酸类代谢物的结合蛋白质  

Identification of the binding proteins of organic acid metabolites by matrix thermal shift assay

作　　者：李柯佳 叶玉莹 张晓磊 周家华 李亚楠 叶明亮[1,2] LI Kejia;YE Yuying;ZHANG Xiaolei;ZHOU Jiahua;LI Yanan;YE Mingliang(CAS Key Laboratory of Separation Science for Analytical Chemistry,Dalian Institute of Chemical Physics,Chinese Academy of Science,Dalian 116023,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院大连化学物理研究所,中国科学院分离分析化学重点实验室,辽宁大连116023 [2]中国科学院大学,北京100049

出　　处：《色谱》2024年第7期702-710,共9页Chinese Journal of Chromatography

基　　金：国家重点研发计划(2021YFA1302601).

摘　　要：有机酸类代谢物在表观遗传、肿瘤发生和发展以及细胞内信号转导等方面发挥着重要作用,对这类代谢物在体内的结合蛋白质进行鉴定,将有助于从分子学层面理解和揭示它们的功能。本研究采用矩阵热转移技术(mTSA),在HeLa细胞裂解液中系统鉴定了3种有机酸类代谢物(琥珀酸、富马酸和乳酸)的结合蛋白质。首先,向细胞裂解液中加入一系列不同浓度的琥珀酸、富马酸或乳酸,在52℃下加热3 min,离心并收集上清蛋白质,进行酶解及后续质谱分析。在数据非依赖性采集(DIA)模式下,本研究在琥珀酸、富马酸和乳酸的mTSA实验中分别鉴定到5870、5744和5816个蛋白质;通过对蛋白质热稳定性的显著性差异值(p)和皮尔森相关系数平方值(R^(2))进行考察,该研究鉴定到了多个高可信度的有机酸类代谢物结合蛋白质。此外本研究发现,虽然富马酸和琥珀酸均能够与α-酮戊二酸依赖的双加氧酶(FTO)结合,但琥珀酸是FTO更强的竞争性抑制剂。除此之外,本研究还鉴定到了两个先前未曾报道过的乳酸结合蛋白质,即鸟氨酸转氨酶(OAT)和3-巯基丙酮酸硫转移酶(MPST),并通过溶剂诱导沉淀技术(SIP)和两种数据检验方法证明了其可信度;同时,OAT和MPST的发现有助于揭示乳酸在氨基酸合成和细胞内氧化还原平衡调节方面的重要作用。Organic acid metabolites exhibit acidic properties.These metabolites serve as intermediates in major carbon metabolic pathways and are involved in several biochemical pathways,including the tricarboxylic acid(TCA)cycle and glycolysis.They also regulate cellular activity and play crucial roles in epigenetics,tumorigenesis,and cellular signal transduction.Knowledge of the binding proteins of organic acid metabolites is crucial for understanding their biological functions.However,identifying the binding proteins of these metabolites has long been a challenging task owing to the transient and weak nature of their interactions.Moreover,traditional methods are unsuitable for the structural modification of the ligands of organic acid metabolites because these metabolites have simple and similar structures.Even minor structural modifications can significantly affect protein interactions.Thermal proteome profiling(TPP)provides a promising avenue for identifying binding proteins without the need for structural modifications.This approach has been successfully applied to the identification of the binding proteins of several metabolites.In this study,we investigated the binding proteins of two TCA cycle intermediates,i.e.,succinate and fumarate,and lactate,an end-product of glycolysis,using the matrix thermal shift assay(mTSA)technique.This technique involves combining single-temperature(52℃)TPP and dose-response curve analysis to identify ligand-binding proteins with high levels of confidence and determine the binding affinity between ligands and proteins.To this end,HeLa cells were lysed,followed by protein desalting to remove endogenous metabolites from the cell lysates.The desalted cell lysates were treated with fumarate or succinate at final concentrations of 0.004,0.04,0.4,and 2 mmol/L in the experimental groups or 2 mmol/L sodium chloride in the control group.Considering that the cellular concentration of lactate can be as high as 2-30 mmol/L,we then applied lactate at final concentrations of 0.2,1,5,10,and 25 mmol/

关 键 词：矩阵热转移技术 有机酸类代谢物 琥珀酸 富马酸 乳酸 结合蛋白质 

分 类 号：O658[理学—分析化学]