title: 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.
authors.affiliation: 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.
authors.forename: Stacie L
authors.initials: SL
authors.lastname: Richardson
authors.affiliation: 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.
authors.forename: Pahul
authors.initials: P
authors.lastname: Hanjra
authors.affiliation: 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.
authors.forename: Gang
authors.initials: G
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Brianna D
authors.initials: BD
authors.lastname: Mackie
authors.affiliation: 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.
authors.forename: Darrell L
authors.initials: DL
authors.lastname: Peterson
authors.affiliation: 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.
authors.forename: Rong
authors.initials: R
authors.lastname: Huang
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D006801:Humans; D007700:Kinetics; D008745:Methylation; D010455:Peptides / Q000032:analysis / Q000378:metabolism*; D011496:Protein Methyltransferases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D010455:Peptides; D011496:Protein Methyltransferases; D000123:Acetyltransferases
keywords: Acetylation; Enzymatic activity assay; MALDI–MS; Methylation; Protein acetyltransferase; Protein methyltransferase