Native chemical ligation approach to sensitively probe tissue acyl-CoA pools.

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James, Andrew M 
Norman, Abigail AI 
Houghton, Jack W 
Prag, Hiran A 
Logan, Angela 

During metabolism, carboxylic acids are often activated by conjugation to the thiol of coenzyme A (CoA). The resulting acyl-CoAs comprise a group of ∼100 thioester-containing metabolites that could modify protein behavior through non-enzymatic N-acylation of lysine residues. However, the importance of many potential acyl modifications remains unclear because antibody-based methods to detect them are unavailable and the in vivo concentrations of their respective acyl-CoAs are poorly characterized. Here, we develop cysteine-triphenylphosphonium (CysTPP), a mass spectrometry probe that uses "native chemical ligation" to sensitively detect the major acyl-CoAs present in vivo through irreversible modification of its amine via a thioester intermediate. Using CysTPP, we show that longer-chain (C13-C22) acyl-CoAs often constitute ∼60% of the acyl-CoA pool in rat tissues. These hydrophobic longer-chain fatty acyl-CoAs have the potential to non-enzymatically modify protein residues.

acyl-CoA, acylation, coenzyme A, cysteine, native chemical ligation, thioester, thiol, triphenylphosphonium, Acyl Coenzyme A, Acylation, Animals, Coenzyme A, Cysteine, Mass Spectrometry, Proteins, Rats
Journal Title
Cell Chem Biol
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Elsevier BV
MRC (MC_UU_00015/3)
Wellcome Trust (220257/Z/20/Z)
Medical Research Council (MC_UU_00015/3)