ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 349 (2006) 112–117 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.10.040 A general liquid chromatography/mass spectroscopy-based assay for detection and quantitation of methyltransferase activity Mary Ellen K. Salyan a , Donna L. Pedicord b , Laurie Bergeron b , Gabe A. Mintier c , Lisa Hunihan d , Kathy Kuit c , Lynn A. Balanda e , Barbara J. Robertson e , John N. Feder c , Ryan Westphal d , Petia A. Shipkova a , Yuval Blat b,¤ a Department of Discovery Analytical Sciences, Bristol–Myers Squibb Co., 311 Pennington-Rocky Hill Rd., Pennington, NJ 08534, USA b Chemical Enzymology, Bristol–Myers Squibb Co., 311 Pennington-Rocky Hill Rd., Pennington, NJ 08534, USA c HT Molecular Biology, Bristol–Myers Squibb Co., 311 Pennington-Rocky Hill Rd., Pennington, NJ 08534, USA d Neuroscience Biology, Bristol–Myers Squibb Co., 5 Research Parkway, Wallingford, CT 06492, USA e Lead Discovery, Bristol–Myers Squibb Co., 5 Research Parkway, Wallingford, CT 06492, USA Received 10 August 2005 Available online 17 November 2005 Abstract Methyltransferases form a large class of enzymes, most of which use S-adenosylmethionine as the methyl donor. In fact, S-adenosylmethio- nine is second only to ATP in the variety of reactions for which it serves as a cofactor. Several methods to measure methyltransferase activity have been described, most of which are applicable only to speciWc enzymes and/or substrates. In this work we describe a sensitive liquid chromatography/mass spectroscopy-based methyltransferase assay. The assay monitors the conversion of S-adenosylmethionine to S-adeno- sylhomocysteine and can be applied to any methyltransferase and substrate of interest. We used the well-characterized enzyme catechol O-methyltransferase to demonstrate that the assay can monitor activity with a variety of substrates, can identify new substrates, and can be used even with crude preparation of enzyme. Furthermore, we demonstrate the utility of the assay for kinetic characterization of enzymatic activity.  2005 Elsevier Inc. All rights reserved. Keywords: Methyltransferase; COMT; S-adenosylmethionine; S-adenosylhomocysteine; LC/MS Methyltransferases are an important class of enzymes. They catalyze the transfer of a methyl group from several diVerent donors to a wide variety of substrates. Substrates include DNA, RNA, proteins, and many diVerent types of small molecules. Methylation can occur on several diVerent atoms including O-, N-, S-, and even C-methylation. The most common methyl donor is S-adenosylmethionine (SAM). 1 SAM-dependent methyltransferases constitute a large group of proteins. In fact, it was estimated that the major class of SAM-dependent methyltranferases, class I, comprises »0.6–1.6% of the genes in a number of prokary- otic and eukaryotic genomes that were evaluated [1]. While SAM-dependent methyltransferases share limited primary sequence homology, they appear to fold into very similar structures. The central core is made of a characteris- tic doubly wound // sandwich structure (see [2] for more details). This core structure provides speciWc contact points for the binding of the cofactor SAM, which include con- served motifs in the G-loop and D-loop and within -strand 4. The methyl transfer reaction proceeds usually through a ternary complex mechanism in which SAM and the substrate are bound to the enzyme at the same time. Methyl transfer occurs via a classical S N 2 mechanism with the methyl acceptor serving as a nucleophile. Following * Corresponding author. Fax: +1 609 818 6935. E-mail address: yuval.blat@bms.com (Y. Blat). 1 Abbreviations used: SAM, S-adenosylmethionine; SAH, S-adenosylho- mocysteine; SIH, S-inosylhomocysteine; COMT, catechol O-methyltrans- ferase; LC/MS, liquid chromatography/mass spectroscopy; MB-COMT, membrane-bound COMT; S-COMT, soluble COMT; DBA, dihydroxy- benzoic acid; 3-MT, 3-methoxytyramine; DTT, dithiothreitol.