FIGURE 4.

Mass spectrometry analysis of the acetylation product made by human NAT8 from S-benzyl-l-cysteine and acetyl CoA. A cell extract from HEK293T cells overexpressing human NAT8 with an N-terminal His₆ tag was incubated for 45 min at 37 °C with 0.25 mm S-benzyl-l-cysteine and 0.2 mm acetyl-CoA. The product of the enzymatic activity was purified from the substrates by reverse phase HPLC and analyzed by mass spectrometry. Tandem mass spectrum of the putative N-acetyl-benzyl-l-cysteine was acquired. The structure of N-acetyl-benzyl-l-cysteine and the assignment of the main fragments are shown.