Sources of S-adenosyl-l-homocysteine background in measuring protein arginine N-methyltransferase activity using tandem mass spectrometry

S-Adenosyl-l-homocysteine (AdoHcy) background signal in reactions with protein arginine N-methyltransferase 1 is investigated using an ultrahigh-performance liquid chromatography tandem mass spectrometry assay that measures AdoHcy. We identify three sources of AdoHcy background: enzymatic automethylation, AdoHcy contamination in commercial S-adenosyl-l-methionine (AdoMet), and nonenzymatic pseudo-first-order formation of AdoHcy from AdoMet. We propose a potential mechanism for the nonenzymatic production of AdoHcy and illustrate strategies for mitigating background AdoHcy that can be applied to any assay.