Poster abstracts
Poster number 27 submitted by Carol Huseby
A liquid chromatography tandem mass spectroscopy approach for quantification of protein methylation stoichiometry
Carol J. Huseby* (Biophysics), Grace L. Cooper* (OSBP), Claire N. Chandler (OSBP), Jean-Christophe Cocuron (Biology and BioDiscovery Institure, University of North Texas), Ana P. Alonso (Biology and BioDiscovery Institure, University of North Texas), Jeff A. Kuret (Biological Chemistry and Pharmacology, Biophysics)
Abstract:
Post-translational modifications are biologically important and wide-spread modulators of protein function. Although methods for detecting the presence of specific modifications are becoming established, approaches for quantifying their mol modification/mol protein stoichiometry are less well developed. Here we introduce a ratiometric, label-free, targeted liquid chromatography tandem mass spectroscopy-based method for estimating Lys and Arg methylation stoichiometry on post-translationally modified proteins. Methylated Lys and Arg were detected with limits of quantification at low fmol and with linearity extending from 20 – 5000 fmol. This level of sensitivity allowed estimation of methylation stoichiometry from microgram quantities of various proteins, including those derived from either recombinant or tissue sources. The method also disaggregated total methylation stoichiometry into its elementary mono-, di-, and tri-methylated residue components. In addition to being compatible with kinetic experiments of protein methylation, the approach is useful for the characterization of methylation states of proteins isolated from cells and tissues.
References:
*equal contribution
Keywords: post-translational modification, protein methylation, mass spectrometry