A direct, ratiometric, and quantitative MALDI–MS assay for protein methyltransferases and acetyltransferases

Abstract

Protein methylation and acetylation play important roles in biological processes, and misregulation of these modifications is involved in various diseases. Therefore, it is critical to understand the activities of the enzymes responsible for these modifications. Herein we describe a sensitive method for ratiometric quantification of methylated and acetylated peptides via MALDI-MS by direct spotting of enzymatic methylation and acetylation reaction mixtures without tedious purification procedures. The quantifiable detection limit for peptides with our method is approximately 10 fmol. This is achieved by increasing the signal-to-noise ratio through the addition of NH4H2PO4 to the matrix solution and reduction of the matrix α-cyanohydroxycinnamic acid concentration to 2 mg/ml. We have demonstrated the application of this method in enzyme kinetic analysis and inhibition studies. The unique feature of this method is the simultaneous quantification of multiple peptide species for investigation of processivity mechanisms. Its wide buffer compatibility makes it possible to be adapted to investigate the activity of any protein methyltransferase or acetyltransferase.

Chemical Biology of Protein N‐Terminal Methyltransferases

“…Cocrystal structures also indicated that NTMT1 had no significant preference for non‐ or monomethylated substrates because they had the same orientation to interact with NTMT1 . This is consistent with comparable kinetic parameters of NTMT1 methylation with both substrates, as well as the distributive mechanism of NTMT1 methylation …”

“…YBR261C and NTMT1/2 are able to methylate nona‐ and hexamer peptides, respectively. In addition, eukaryotic protein NTMTs share an X‐P‐K/R motif.…”

“…Mutant H140A lost the catalytic activity, but retained binding affinity to the peptide substrate . NTMT1 is known to be a trimethylase that catalyzes mono‐, di‐, and trimethylation . During the process of multiple methylations, the substrate can be released and rebind to NTMT1, which proceeds through a distributive mechanism for multiple methylations …”

“…NTMT2i sp redominantly expressed in heart and skeletal muscle tissues, [28] whichs uggesting the possibility of its role in at issue-specific context. YBR261C and NTMT1/2 are able to methylate nona-and hexamer peptides, [11,26,[31][32][33] respectively.I na ddition, eukaryotic protein NTMTss hare an X-P-K/R motif. Therefore, it is reasonable to speculate that an N-terminal linear sequence is sufficient for their recognition.…”

Facile synthesis of SAM–peptide conjugates through alkyl linkers targeting protein N-terminal methyltransferase 1

“…At 30 μM, it did not show any significant inhibition of either G9a or PRMT1. We also examined how compound 1c affects the progression of α- N -amine methylation at 5 μM by MALDI-MS. 9,24,41 Triplicate samples of RCC1-10 peptide (SPKRIAKRRS) along with compound 1c were subjected to NTMT1 methylation assays. Following these assays, samples were analyzed at 20 min to monitor the methylation progression.…”