A liquid chromatography tandem mass spectroscopy approach for quantification of protein methylation stoichiometry

Grace L. Cooper, Carol J. Huseby, Claire N. Chandler, Jean Christophe Cocuron, Ana P. Alonso, Jeff Kuret

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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 to 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 will be especially useful for characterizing methylation states of proteins isolated from cells and tissues.

Original languageEnglish (US)
Pages (from-to)72-77
Number of pages6
JournalAnalytical Biochemistry
Volume545
DOIs
StatePublished - Mar 15 2018
Externally publishedYes

Keywords

  • Label-free
  • Mass spectrometry
  • Post-translational modification
  • Protein methylation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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