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.
Lakowski
Ted M
TM
Division of Biomolecular and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
Frankel
Adam
A
D016477:Artifacts; D002853:Chromatography, Liquid; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D008745:Methylation; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000032:analysis* / Q000737:chemistry; D012436:S-Adenosylmethionine / Q000737:chemistry; D053719:Tandem Mass Spectrometry
D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D011484:Protein-Arginine N-Methyltransferases