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

doi:10.3724/SP.J.1123.2023.07002

Abstract

Abstract:

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.

Key words: matrix thermal shift assay (mTSA), organic acid metabolites, succinate, fumarate, lactate, binding proteins

CLC Number:

O658

LI Kejia, YE Yuying, ZHANG Xiaolei, ZHOU Jiahua, LI Yanan, YE Mingliang. Identification of the binding proteins of organic acid metabolites by matrix thermal shift assay[J]. Chinese Journal of Chromatography, 2024, 42(7): 702-710.

Figures/Tables 4

Fig. 1 Evaluation of the depth of identification and quantitative repeatability of succinate, fumarate and lactate in the matrix thermal shift assay (mTSA) protein dataset R2: square of Pearson’s correlation coefficient.

Fig. 2 Identification and affinity calculation of known binding proteins of succinate, fumarate and lactate a-c. volcano plot visualization generated by comparing protein intensities between 2 mmol/L succinate, 2 mmol/L fumarate and 25 mmol/L lactate treatment and vehicle treatment, respectively. A threshold of p<0.001 (represented by the dashed grey line) was used to determine potential binding proteins. d-f. mTSA experiment for succinate, fumarate and lactate, respectively; scatter plot of -log10 p (maximum concentration of the organic acid metabolite vs vehicle) and R2 to the sigmoid trend of the dose-response curve. The determined potential binding proteins were labeled in red. g, h. dose-response curves for fumarate hydratase (FH) and α-ketoglutarate-dependent dioxygenase (FTO) by treatment with different concentrations of fumarate, respectively. i. dose-response curves for FTO by treatment with different concentrations of succinate. FC: fold change; p: p-value; OAT: ornithine aminotransferase; MPST: 3-mercaptopyruvate sulfurtransferase; LDHA: L-lactate dehydrogenase A chain; LDHB: L-lactate dehydrogenase B chain; EC50: half effective ligand concentration.

Fig. 3 Validation of unknown lactate-binding proteins a. assessment of quantification reproducibility of the proteins in the solvent-induced precipitation (SIP) experiment by R2; b. volcano plot visualization generated by comparing protein intensities between 25 mmol/L lactate treatment and vehicle treatment. OAT and MPST that identified as potential lactate-binding proteins in the mTSA experiment were labeled in red.

Fig. 4 Multiple data test for MPST a, b. volcano plot visualization generated by comparing protein intensities between 10 mmol/L lactate treatment and vehicle treatment, 25 mmol/L lactate treatment and vehicle treatment, respectively. The top two proteins with the lowest p were colored in red. c. volcano plot visualization generated by comparing peptide intensities between 25 mmol/L lactate treatment and vehicle treatment. The peptides of MPST were colored in red. A p threshold of 0.005 (indicated by a dashed red line) was applied to determine significant changed peptides.

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