Global Proteomics Analysis of Protein Lysine Methylation

Xing‐Jun Cao1, Benjamin A. Garcia1

1 Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 24.8
DOI:  10.1002/cpps.16
Online Posting Date:  November, 2016
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Lysine methylation is a common protein post‐translational modification dynamically mediated by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs). Beyond histone proteins, lysine methylation on non‐histone proteins plays a substantial role in a variety of functions in cells and is closely associated with diseases such as cancer. A large body of evidence indicates that the dysregulation of some PKMTs leads to tumorigenesis via their non‐histone substrates. However, most studies on other PKMTs have made slow progress owing to the lack of approaches for extensive screening of lysine methylation sites. However, recently, there has been a series of publications to perform large‐scale analysis of protein lysine methylation. In this unit, we introduce a protocol for the global analysis of protein lysine methylation in cells by means of immunoaffinity enrichment and mass spectrometry. © 2016 by John Wiley & Sons, Inc.

Keywords: immunoaffinity enrichment; methylation; mass spectrometry; non‐histone; post‐translational modification; proteomics

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Table of Contents

  • Introduction
  • Basic Protocol 1: Large‐Scale Analysis of Protein Lysine Methylation in Cells
  • Support Protocol 1: Antisera Generation and Affinity Purification of Antibody
  • Support Protocol 2: Preparation of Capillary Columns with Integrated Emitter Tips
  • Reagents And Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Large‐Scale Analysis of Protein Lysine Methylation in Cells

  • 0.25% trypsin‐EDTA (Gibco)
  • 1 M Tris·HCl, pH 8.3 (see recipe)
  • Urea lysis buffer (see recipe)
  • Bradford protein assay solution (see unit 3.4; Olson and Markwell, )
  • 1 M DTT stock solution (see recipe)
  • 1 M iodoacetamide (IAA) solution (see recipe)
  • Trypsin
  • Trifluoroacetic acid (TFA)
  • Methanol, HPLC grade
  • Acetic acid
  • Formic acid (FA)
  • Acetonitrile
  • SCX Buffer A (see recipe)
  • SCX Buffer B (see recipe)
  • Protein A Mag sepharose (GE Healthcare)
  • Pan anti‐monomethyl lysine antibody (see protocol 2)
  • Saturated Na 2HPO 4 in PBS (see recipe)
  • 0.8 M NaCl in PBS
  • Reverse‐phase HPLC Buffer A (see recipe)
  • Reverse‐phase HPLC Buffer B (see recipe)
  • pH indicator strip (pH range 1 to 14)
  • Centrifuge
  • Sep‐Pak C18 cartridges (Waters)
  • PolySULFOETHYL A column (9.4 mm I.D. × 250 mm, PolyLC)
  • Empore C18 disk (3M)
  • Reverse‐phase HPLC column (see protocol 3)
  • Probe sonicator
  • QIAvac vacuum manifold
  • HPLC with UV‐detector
  • SpeedVac concentrator
  • Magnetic rack
  • Tube rotator

Support Protocol 1: Antisera Generation and Affinity Purification of Antibody

  Additional Materials (also see protocol 1Basic Protocol)
  • Tris(2‐carboxyethyl) phosphine (TCEP)
  • 1 M NaCl
  • 50 mM L‐cysteine (see recipe)
  • SulfoLink coupling resin
  • Resin coupling buffer (see recipe)
  • Sodium azide
  • Centrifuge
  • Econo‐Pac chromatography columns
  • Tube rotator

Support Protocol 2: Preparation of Capillary Columns with Integrated Emitter Tips

  • Methanol
  • Acetonitrile
  • HF acid
  • 0.2 M ammonium formate
  • Reprosil‐Pur C18‐AQ resin (3 µm; Dr. Maisch)
  • BSA digests
  • Reverse‐phase HPLC Buffer A (see recipe)
  • 360 μm O.D. × 75 μm I.D. fused silica tubing
  • Lighter
  • Lamp
  • Laser puller P‐2000
  • Helium tank and its accessories
  • Pressure bomb (Nanobaume, cat. no. SP‐400)
  • 2‐ml HPLC glass vial
  • Stirrer
  • Microscope
  • HPLC instrument (e.g., Easy nLC 1000 HPLC)
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Literature Cited

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