An optimized method using peptide arrays for the identification of in vitro substrates of lysine methyltransferase enzymes

Elyn M Rowe,Kyle K Biggar

doi:10.1016/j.mex.2018.01.012

Abstract

While a number of post-translational modifications (PTM), such as phosphorylation and ubiquitination, have been extensively studied, lysine methylation is emerging as an important PTM with implications in a growing number of diverse cellular processes. To date, there are approximately 5000 identified methylation sites on non-histone proteins, and as the methyllysine proteome expands it becomes important to identify the lysine methyltransferase enzymes responsible for each methylation event. The use of peptide SPOT methylation assay has proven to be a useful in the identification and validation of novel substrates for lysine methyltransferase enzymes as it uses a weak beta emitter coupled with fluorography to detect methylation events. The method described in this paper provides improvements to the typical protocol for this assay, as a highly sensitive tritium assay can be developed with less radioactivity than previously described. This protocol provides an inexpensive alternative to weak beta signal enhancer sprays and washes for use in lysine methylation peptide SPOT arrays, and a simple open-source method for array quantification.

Highlights

While effective weak beta emitter enhancer sprays and washes are commercially available, the method described below provides an inexpensive alternative for producing clear images of radioactively labelled peptide arrays using 2,5diphenyloxazole (DPO)
This method provides the optimized amounts of radioactivity needed to reduce the potentially high costs of in vitro methylation assays, and is able to use a lower concentration of radioactivity than previously reported [4]
The described method was used to effectively two known and well-studied non-histone substrates of SMYD3; a KMT that has been found to be over-expressed in many types of cancer [7]

Summary

Method Article

Optimized concentration of radioactivity for a safer and more cost-efficient method Effective enhancer alternative described for expensive tritium enhancer sprays for array-based fluorometry assays Open-source array quantification for experimental analysis It is well-established that lysine methyltransferase (KMT) enzymes play a role in the methylation of histones and in effect, the regulation of gene expression in the nucleus. While effective weak beta emitter enhancer sprays and washes are commercially available, the method described below provides an inexpensive alternative for producing clear images of radioactively labelled peptide arrays using 2,5diphenyloxazole (DPO) This method provides the optimized amounts of radioactivity needed to reduce the potentially high costs of in vitro methylation assays, and is able to use a lower concentration of radioactivity than previously reported [4]

Method details

Procedure

Method validation