Analysis of Histone Modifications by Mass Spectrometry

Ana Villar‐Garea1, Lars Israel2, Axel Imhof1

1 Histone Modifications Group, Biomedical Center of the Ludwig Maximilians University of Munich, Germany, 2 Protein Analysis Unit, Biomedical Center of the Ludwig Maximilians University of Munich, Germany
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.10
DOI:  10.1002/0471140864.ps1410s51
Online Posting Date:  February, 2008
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Abstract

Histone N‐termini undergo diverse post‐translational modifications that significantly extend the information potential of the genetic code. Moreover, they appear to mark specific chromatin regions, modulating epigenetic control, lineage commitment, and overall function of chromosomes. It is widely accepted that histone modifications affect chromatin function, but the exact mechanisms of how modifications on histone tails and specific combinations of modifications are generated, and how they cross‐talk with one another, is still enigmatic. Mass spectrometry is ideal for the analysis of histone modifications and is becoming the gold standard for histone post‐translational modification analysis since it allows the quantification of modifications and combinations. This unit describes how high‐resolution mass spectrometry can be used to study histone post‐translational modifications. Curr. Protoc. Protein Sci. 51:14.10.1‐14.10.14. © 2008 by John Wiley & Sons, Inc.

Keywords: histone modifications; lysine acetylation; lysine methylation; mass spectrometry; tandem MS; chemical derivatization of amino acids

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Acid Extraction of Histones
  • Basic Protocol 2: Histone Acylation and Digestion with Trypsin
  • Basic Protocol 3: MALDI‐TOF Analysis of Trypsin‐Digested Histones
  • Basic Protocol 4: Analysis of Modified Histone Peptides by Tandem MS (MS/MS)
  • Support Protocol 1: Formic Acid Extraction of Peptides After In‐Gel Digestion
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Acid Extraction of Histones

  Materials
  • Eukaryotic cells or nuclei preparation
  • 0.25 M HCl
  • 0.1 M acetic acid containing 1 mM dithiothreitol (DTT; added fresh just before use)
  • 20 mM Tris·Cl pH 6.8 ( appendix 2E) containing 1 mM DTT (or other reducing agent, added fresh just before use)
  • Thin‐tipped pipet or Dounce homogenizer
  • Shaking incubator or rotator, 4°C
  • Dialysis bags (MWCO 6 to 8 kDa)

Basic Protocol 2: Histone Acylation and Digestion with Trypsin

  Materials
  • Histone samples separated by SDS‐PAGE (see )
  • 10 mM ammonium bicarbonate
  • Acetonitrile (HPLC grade)/50 mM ammonium bicarbonate
  • Acetonitrile, HPLC grade
  • Propanoic anhydride or acetic anhydride‐d6
  • 0.1 M ammonium bicarbonate
  • 1 M ammonium bicarbonate
  • 50 mM ammonium bicarbonate, ice cold
  • 0.2 µg/µl sequencing grade trypsin, prepared according to the manufacturer's instructions
  • Scalpel
  • Shaking incubator, 37°C
  • pH‐indicator paper

Basic Protocol 3: MALDI‐TOF Analysis of Trypsin‐Digested Histones

  Materials
  • Acetonitrile, HPLC grade
  • 0.6% (v/v) trifluoroacetic acid (TFA), spectroscopy grade
  • α‐cyano‐4‐hydroxycinnamic acid (CHCA; Sigma‐Aldrich), mass spectrometry grade
  • Trypsin digest ( protocol 2) containing ∼0.1 µg of histone
  • SCX (strong cation exchange resin) or µC18 (reversed‐phase resin) ZipTips (Millipore)
  • Wetting, equilibrating, and washing solutions: prepared according to the ZipTip manufacturer's instructions or as indicated
  • Washing solution for SCX tips: 0.1% TFA (without methanol)
  • Eluting solution for SCX tips: 5% (v/v) ammonium hydroxide/30% (v/v) methanol (HPLC grade)
  • Bath sonicator
  • MALDI target plate
  • Additional reagents and equipment for performing MALDI‐TOF spectrometry (unit 16.2)

Basic Protocol 4: Analysis of Modified Histone Peptides by Tandem MS (MS/MS)

  Materials
  • Trypsin digest ( protocol 2) containing ∼1 µg of histone
  • Formic acid, analysis grade
  • µC18 ZipTips (Millipore)
  • 50% (v/v) methanol/0.1% (v/v) formic acid
  • Ammonium hydroxide
  • GELoader tip (Eppendorf, or similar with extended tips)
  • Needle holder (see Fig. ), optional
  • Nano‐electrospray capillaries, 1 µm internal diameter (e.g., Protana)
  • Additional reagents and equipment for desalting peptides ( protocol 3) and analyzing peptides using nanospray techniques and mass spectroscopy (unit 16.8)

Support Protocol 1: Formic Acid Extraction of Peptides After In‐Gel Digestion

  Material
  • Trypsin digest supernatant from gel and gel pieces ( protocol 2)
  • 50 mM ammonium bicarbonate
  • 50% (v/v) acetonitrile/25 mM ammonium bicarbonate
  • 5% (v/v) formic acid
  • 50% (v/v) acetonitrile/2.5% (v/v) formic acid
  • Acetonitrile, HPLC grade
  • 0.1% (v/v) trifluoroacetic acid (TFA), spectroscopy grade
  • 0.5‐ml low protein‐binding plastic tube
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Figures

Videos

Literature Cited

Literature Cited
   Bonaldi, T., Imhof, A., and Regula, J.T. 2004a. A combination of different mass spectrometry techniques for the analysis of dynamic changes of histone modifications. Proteomics 4:1382‐1396
   Bonaldi, T., Regula, J.T., and Imhof, A. 2004b. The use of mass spectrometry for the analysis of histone modifications. In Methods in Enzymology, Vol. 377: Chromatin and Chromatin Remodeling Enzymes (C.D. Allis and C. Wu, eds.) pp. 111‐130. Elsevier, London.
   Chang, L., Ryan, C.A., Schneider, C.A., and Annunziato, A.T. 1999. Preparation/analysis of chromatin replicated in vivo and in isolated nuclei. Methods Enzymol. 304:76‐99.
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