有机酸代谢物表现出酸性。这些代谢物作为主要碳代谢途径的中间体，并参与多种生化途径，包括三羧酸（TCA）循环和糖酵解。它们还调节细胞活动，并在表观遗传学、肿瘤发生和细胞信号转导中发挥关键作用。了解有机酸代谢物的结合蛋白对于理解其生物学功能至关重要。然而，由于这些代谢物相互作用的短暂性和弱性，识别这些代谢物的结合蛋白长期以来一直是一项具有挑战性的任务。此外，传统方法不适合有机酸代谢物配体的结构修饰，因为这些代谢物结构简单且相似。即使是微小的结构修饰也会显着影响蛋白质相互作用。热蛋白质组分析 (TPP) 为鉴定结合蛋白提供了一种有前途的途径，而无需进行结构修饰。该方法已成功应用于多种代谢物结合蛋白的鉴定。在本研究中，我们使用基质热位移测定 (mTSA) 技术研究了两种 TCA 循环中间体（即琥珀酸和富马酸）以及乳酸（糖酵解的终产物）的结合蛋白。该技术结合单温（52℃）TPP和剂量反应曲线分析，以高置信度鉴定配体结合蛋白，并确定配体和蛋白质之间的结合亲和力。为此，裂解 HeLa 细胞，然后进行蛋白质脱盐，以去除细胞裂解物中的内源代谢物。实验组用终浓度为0.004、0.04、0.4和2 mmol/L的富马酸盐或琥珀酸盐处理脱盐的细胞裂解液，对照组用终浓度为2 mmol/L的氯化钠处理。考虑到细胞内乳酸浓度可高达2-30 mmol/L，因此实验组应用终浓度为0.2、1、5、10和25 mmol/L的乳酸或25 mmol/L钠对照组的氯化物。使用高灵敏度质谱结合数据独立采集 (DIA) 定量，我们分别在琥珀酸盐、富马酸盐和乳酸 mTSA 实验中定量了 5870、5744 和 5816 个蛋白质。通过设定严格的截止值（即蛋白质热稳定性变化的显着性（p值）<0.001和剂量反应曲线拟合的质量（皮尔逊相关系数的平方，R2）>0.95），多种结合蛋白这些来自背景蛋白质的有机酸代谢物被可靠地测定。鉴定了几种已知的结合蛋白，特别是富马酸水合酶（FH）作为富马酸的结合蛋白，以及α-酮戊二酸依赖性双加氧酶（FTO）作为富马酸和琥珀酸的结合蛋白。此外，还获得了这些代谢物与其结合蛋白之间相互作用的亲和力数据，这与文献中报道的数据非常匹配。有趣的是，参与氨基酸生物合成的鸟氨酸转氨酶（OAT）和在细胞中充当抗氧化剂的3-巯基丙酮酸硫转移酶（MPST）被鉴定为乳酸结合蛋白。随后，我们实验室开发了一种正交测定技术，即溶剂诱导沉淀（SIP）技术，用于验证 mTSA 结果。 SIP 将 OAT 确定为首要候选目标，验证了基于 mTSA 的发现，即 OAT 是一种新型乳酸结合蛋白。尽管SIP未将MPST鉴定为乳酸结合蛋白，但在10或25 mmol/L乳酸的mTSA实验中对MPST的统计分析表明MPST是具有高置信度的乳酸结合蛋白。肽水平经验贝叶斯 t 检验与 Fisher 精确检验相结合也支持 MPST 是乳酸结合蛋白的结论。乳酸在结构上与丙酮酸相似，丙酮酸是已知的 MPST 结合蛋白。因此，假设乳酸可能会占据 MPST 上丙酮酸的结合位点。总体而言，针对乳酸鉴定的新型结合蛋白表明它们可能参与氨基酸合成和氧化还原平衡调节。

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/L in the experimental groups or 25 mmol/L sodium chloride in the control group. Using high-sensitivity mass spectrometry coupled with data-independent acquisition (DIA) quantification, we quantified 5870, 5744, and 5816 proteins in succinate, fumarate, and lactate mTSA experiments, respectively. By setting stringent cut-off values (i.e., significance of changes in protein thermal stability (p-value)<0.001 and quality of the dose-response curve fitting (square of Pearson's correlation coefficient, R2)>0.95), multiple binding proteins for these organic acid metabolites from background proteins were confidently determined. Several known binding proteins were identified, notably fumarate hydratase (FH) as a binding protein for fumarate, and α-ketoglutarate-dependent dioxygenase (FTO) as a binding protein for both fumarate and succinate. Additionally, the affinity data for the interactions between these metabolites and their binding proteins were obtained, which closely matched those reported in the literature. Interestingly, ornithine aminotransferase (OAT), which is involved in amino acid biosynthesis, and 3-mercaptopyruvate sulfurtransferase (MPST), which acts as an antioxidant in cells, were identified as lactate-binding proteins. Subsequently, an orthogonal assay technique developed in our laboratory, the solvent-induced precipitation (SIP) technique, was used to validate the mTSA results. SIP identified OAT as the top target candidate, validating the mTSA-based finding that OAT is a novel lactate-binding protein. Although MPST was not identified as a lactate-binding protein by SIP, statistical analysis of MPST in the mTSA experiments with 10 or 25 mmol/L lactate revealed that MPST is a lactate-binding protein with a high level of confidence. Peptide-level empirical Bayes t-tests combined with Fisher's exact test also supported the conclusion that MPST is a lactate-binding protein. Lactate is structurally similar to pyruvate, the known binding protein of MPST. Therefore, assuming that lactate could potentially occupy the binding site of pyruvate on MPST. Overall, the novel binding proteins identified for lactate suggest their potential involvement in amino acid synthesis and redox balance regulation.