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Mass Spectrometry Analysis of Lysine Acetylation in Citrate Synthase Pulldown Samples

ABSTRACT: This dataset contains raw mass spectrometry (MS) data generated from immunoprecipitation experiments aimed at identifying acetylation sites on proteins interacting with citrate synthase (CS) in mouse cardiac cells. Two groups of cells were analyzed: negative control (NC) and overexpressing cells, both subjected to experimental treatment prior to sample collection.

INSTRUMENT(S): Orbitrap Elite LC-MS/MS

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Yin Deling

PROVIDER: MSV000098986 | MassIVE | Sun Aug 31 01:07:00 BST 2025

REPOSITORIES: MassIVE

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Project description:The cytokines tumor necrosis factor ligand superfamily member 11 (TNFSF11; also known as RANKL) and macrophage colony-stimulating factor 1 receptor (M-CSF) differentiate macrophages into bone-resorbing osteoclasts via a process characterised by changes in metabolic activity that support energy-consuming processes such as cell fusion or bone resorption. Treatment with RANKL triggers a phenotype of accelerated metabolism with enhanced glycolysis and an initial disruption of the tricarboxylic acid cycle (TCA) cycle through increased expression of the enzyme aconitate decarboxylase (ACOD1). which results in an upregulation of intracellular succinate levels. Succinate then causes post-translational succinylation of lysine residues. Interestingly, ACOD1 as an inducer of protein succinylation and the desuccinylase NAD-dependent protein deacylase sirtuin-5, mitochondrial (SIRT5) are regulated differentially and the initially high expression of ACOD1 decreases towards the end of differentiation, whereas SIRT5 levels increase. To mimic the effect of protein succinylation, diethyl succinate or a SIRT5 inhibitor were added to differentiating osteoclasts, which reduced the formation of large osteoclasts, showing its relevance for successful osteoclastogenesis. To identify proteins succinylated after RANKL treatment, we used an immunoaffinity-based liquid chromatography–tandem mass spectrometry (LC-MS/MS) approach. Most lysine succinylated proteins were metabolic enzymes localised in the mitochondria. Citrate synthase, the enzyme catalysing the first reaction of the TCA cycle, showed a notable difference in succinylation levels before and after RANKL stimulation, with succinylation detected exclusively in stimulated cells. Immunoprecipitation assays confirmed citrate synthase succinylation. Using whole cell extracts, we observed that RANKL treatment decreased CS activity in a concentration-dependent manner. This suggests that CS could be a critical factor in the context of energy production during osteoclastogenesis and that protein succinylation helps to modulate the differentiation program of osteoclasts.

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Mass Spectrometry Analysis of Lysine Acetylation in Citrate Synthase Pulldown Samples

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