Approaches to measuring the activities of protein arginine N-methyltransferases

Abstract

Despite the emerging importance of protein arginine N-methyltransferase (PRMT) activity in regulating cellular processes, only a limited number of PRMT assays have been developed. Here, we compare several qualitative and quantitative methods that we use for measuring PRMT activity. Gel-based methods allow for the simultaneous detection of methyl transfer activity on multiple substrates, but require signals well above background in order to generate reliable data for quantitation, which can be challenging with low activity PRMTs or substrates that are poor methyl-acceptors. Techniques that measure S-adenosyl-L-homocysteine (AdoHcy) product formation suffer from a background caused by PRMT automethylation and the spontaneous formation of AdoHcy from S-adenosyl-L-methionine (AdoMet). However, when this background is controlled, this approach is useful for product inhibition studies. Methods that detect methylated arginines derived from acid hydrolysis of PRMT reaction samples can determine the absolute amounts of ω-NG-monomethylarginine (MMA), asymmetric ω-NG,NG-dimethylarginine (aDMA) or symmetric ω-NG,N′G-dimethylarginine (sDMA) to quantify PRMT activity. We describe separation methods of these methylated arginine derivatives by thin layer, reverse phase, or cation exchange chromatography, and quantification by radioactivity or mass spectrometry. The latter approach is advantageous because it does not require radiolabelled samples for detection, and activity is readily quantified with commercially available standards.

Arginine methylation in yeast proteins during stationary-phase growth and heat shock

“…A potential explanation is that when specific RMT enzymes are knocked out, additional narrow substrate specificity enzymes compensate by broadening substrate specificity. This phenomenon has been observed previously in PRMT1 knockout cells (Dhar et al 2013) and comports with our in vitro data that suggest that PRMTs will methylate histones that they do not normally prefer if they are the only available substrates (Lakowski et al 2010b). These results suggest that enzymes other than Hsl7p may be producing sDMA in yeast.…”

“…Figure 2b displays the MS 2 of sDMA (top) and the MS 3 derived from the primary product ions 158.1 m/z (middle) and 172.0 m/z (bottom). As observed previously, the MS 2 for sDMA and aDMA are similar except for the unique product ions 46 m/z for aDMA and 172.1 m/z for sDMA (Rappsilber et al 2003;Brame et al 2004;Gehrig et al 2004;Lakowski and Frankel 2009;Lakowski et al 2010b;Pak et al 2011). The MS 3 spectrum of the primary product ion 172.2 m/z is diagnostic for sDMA (Fig.…”

Formulating a fluorogenic assay to evaluate S-adenosyl-L-methionine analogues as protein methyltransferase cofactors

“…Given low catalytic efficiency for most PMTs ( k cat < 1 min −1 ), 37, 41, 45, 46 established PMT-activity assays mainly rely on radioactive SAM, antibodies, mass spectrometry or coupling enzymes to quantify reaction products or byproduct (methylated products or SAH), rather than the consumption of substrates or the SAM cofactor. 30, 43, 51 In the reactions with native or mutated PMTs and SAM analogues, SAH production is the solely-shared character regardless of PMTs, PMT substrates and the SAM analogues (Figure 1). This feature therefore presents SAH quantification an amenable and potentially generalizable approach to probe the reactivity of PMTs and their variants on structurally-diverse substrates and SAM analogues.…”

Methods Applied to the Study of Protein Arginine Methylation

“…A few techniques that are central to the analysis of arginine methylation have not been discussed here. These include amino acid hydrolysis, and the subsequent analysis of methylated amino acid using a variety of different chromatography approaches to evaluate types and levels of arginine methylation, and a detailed description of these detection methods can be found in the following reference (Lakowski, Zurita-Lopez, Clarke, & Frankel, 2010). We do not address the mass spectrometry techniques that are used to distinguish between SDMA and ADMA marks (Brame, Moran, & McBroom-Cerajewski, 2004;Wang et al, 2009).…”