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Anal Biochem. 2015 Jun 1;478:59-64. doi: 10.1016/j.ab.2015.03.007. Epub 2015 Mar 14.

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

Author information

1
Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23219, USA; Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23219, USA.
2
Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23219, USA; Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23219, USA.
3
Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23219, USA; Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23219, USA. Electronic address: rhuang@vcu.edu.

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.

KEYWORDS:

Acetylation; Enzymatic activity assay; MALDI–MS; Methylation; Protein acetyltransferase; Protein methyltransferase

PMID:
25778392
PMCID:
PMC4855292
DOI:
10.1016/j.ab.2015.03.007
[Indexed for MEDLINE]
Free PMC Article

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