Comparison of a High-Throughput Mass Spectrometry Method and Radioactive Filter Binding to Assay the Protein Methyltransferase PRMT5

Assay Drug Dev Technol. 2015 May;13(4):235-40. doi: 10.1089/adt.2015.640.

Abstract

Conformational remodeling of chromatin in cells is known to alter gene expression. The histone code hypothesis postulates that multiple modifications present on histone tails can regulate gene expression both through direct effects on chromatin compaction as well as through recruitment of unique complexes that signal specific downstream functions. Histone methylation is an important component of the histone code, and the dysregulation of histone methylation in disease makes methyltransferases and demethylases viable targets for drug discovery. We developed a biochemical assay platform, which takes advantage of the fact that protein methyltransferases (PMTs) all utilize the cofactor S-Adenosyl-L-methionine (SAM) as the methyl donor. The platform utilizes the High-throughput Mass Spectrometry (MS) technology to measure SAM and the S-Adenosyl-L-homocysteine product in a label-free manner. The platform has all the advantages of a label-free system coupled with the benefit of substrate agnostic measurements making it an ideal setup for PMT biochemical studies and drug discovery. In addition, MS is ideally suited for detecting multiple modification events within the same substrate. The ability to adjust the detection to monitor the methyl acceptor product allows for real-time measurements of multiple product species simultaneously, a distinct advantage over other commonly used assay formats.

Publication types

  • Comparative Study

MeSH terms

  • Binding Sites
  • Enzyme Assays / methods*
  • High-Throughput Screening Assays*
  • Humans
  • Mass Spectrometry*
  • Protein-Arginine N-Methyltransferases / analysis*
  • Protein-Arginine N-Methyltransferases / metabolism
  • Radiometry / methods*

Substances

  • PRMT5 protein, human
  • Protein-Arginine N-Methyltransferases