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

Abstract

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 potentially 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 utilizes ‘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.