One-pot shotgun quantitative mass spectrometry characterization of histones

Mariana D. Plazas-Mayorca, Barry M. Zee, Nicolas L. Young, Ian M. Fingerman, Gary LeRoy, Scott D. Briggs, Benjamin A. Garcia

Research output: Contribution to journalArticlepeer-review

113 Scopus citations

Abstract

Despite increasing applications of mass spectrometry (MS) to characterize post-translational modifications (PTMs) on histone proteins, most existing protocols are not properly suited to robustly measure them in a high-throughput quantitative manner. In this work, we expand on current protocols and describe improved methods for quantitative Bottom Up characterization of histones and their PTMs with comparable sensitivity but much higher throughput than standard MS approaches. This is accomplished by first bypassing off-line fractionation of histone proteins and working directly with total histones from a typical nuclei acid extraction. Next, using a chemical derivatization procedure that is combined with stable-isotope labeling in a two-step process, we can quantitatively compare samples using nanoLC-MS/MS. We show that our method can successfully detect 17 combined H2A/H2B variants and over 25 combined histone H3 and H4 PTMs in a single MS experiment. We test our method by quantifying differentially expressed histone PTMs from wild-type yeast and a methyltransferase knockout strain. This improved methodology establishes that time and sample consuming off-line HPLC or SDS-PAGE purification of individual histone variants prior to MS interrogation as commonly performed is not strictly required. Our protocol significantly streamlines the analysis of histone PTMs and will allow for studies of differentially expressed PTMs between multiple samples during biologically relevant processes in a rapid and quantitative fashion.

Original languageEnglish
Pages (from-to)5367-5374
Number of pages8
JournalJournal of Proteome Research
Volume8
Issue number11
DOIs
StatePublished - Nov 6 2009

Keywords

  • Electron transfer dissociation
  • Histone
  • Mass spectrometry
  • Post-translational modification
  • Proteomics
  • Quantitative
  • Stable isotope labeling

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