Large‐Scale Identification of the Arginine Methylome by Mass Spectrometry

Kathrine B. Sylvestersen1, Michael L. Nielsen1

1 The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences, Copenhagen
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 24.7
DOI:  10.1002/0471140864.ps2407s82
Online Posting Date:  November, 2015
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The attachment of one or more methylation groups to the side chain of arginine residues is a regulatory mechanism for cellular proteins. Recent advances in mass spectrometry−based characterization allow comprehensive identification of arginine methylation sites by peptide‐level enrichment strategies. Described in this unit is a 4‐day protocol for enrichment of arginine‐methylated peptides and subsequent identification of thousands of distinct sites by mass spectrometry. Specifically, the protocol explains step‐by‐step sample preparation, enrichment using commercially available antibodies, prefractionation using strong cation exchange, and identification using liquid chromatography coupled to tandem mass spectrometry. A strategy for relative quantification is described using stable isotope labeling by amino acids in cell culture (SILAC). Approaches for analysis of arginine methylation site occupancy are also discussed. Collectively, the unit describes the essential parameters required for a successful and comprehensive experiment detailing the arginine methylome. © 2015 by John Wiley & Sons, Inc.

Keywords: arginine methylation; quantitative mass spectrometry; SILAC; post‐translational modification

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Global Antibody Enrichment and Ms Identification of Arginine‐ Methylated Peptides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Global Antibody Enrichment and Ms Identification of Arginine‐ Methylated Peptides

  • Cells of interest
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010‐023), 4°C
  • Modified RIPA lysis buffer (see recipe)
  • Bradford reagent (e.g., Bio‐Rad Quick Start Bradford 1× Dye Reagent)
  • Acetone (Merck, cat. no. 1.00020.2500), ice cold
  • Denaturation buffer, pH 8.0 (see recipe)
  • Reduction buffer (see recipe)
  • Alkylation buffer (see recipe)
  • Lys‐C protease (Wako Chemicals, cat no. 129‐02541)
  • Digestion buffer: 50 mM ammonium bicarbonate, pH 8.0
  • Trypsin (sequencing grade, modified, 1 mg/vial; Promega, cat. no. V511X)
  • 0.1%, 0.15%, and 10% (v/v) trifluoroacetic acid (TFA)
  • 50% and 70% (v/v) acetonitrile (Merck, cat. no. 1.00030.2500)
  • 10× and 1× immunoprecipitation buffer (PTMScan IAP Buffer from kit or see recipe)
  • PTMScan methylated arginine kits (Cell Signaling Technology):
  • Mono‐Methyl Arginine Motif [mme‐RG] kit (cat. no. 12235)
  • Asymmetric Di‐Methyl Arginine Motif [adme‐R] kit (cat. no. 13474)
  • Symmetric Di‐Methyl Arginine Motif [sdme‐R] kit (cat. no. 13563)
  • Methanol (Merck, cat. no. 1.06007.2500)
  • SCX conditioning buffer: 20% acetonitrile/0.1% TFA
  • SCX buffer (see recipe) at pH 4, 4.5, 5.5, 6.5, 8.0, and 11
  • Buffer A: 0.1% (v/v) acetic acid
  • Buffer B: 80% acetonitrile/0.1% acetic acid
  • StageTip elution solvent 1: 40% acetonitrile, 0.1% formic acid
  • StageTip elution solvent 2: 60% acetonitrile, 0.1% formic acid
  • Culture dishes (e.g., 150‐ or 245‐mm)
  • Cell scraper
  • 50‐ and 15‐ml Falcon tubes
  • Probe sonicator
  • Thermomixer
  • Sep‐Pak solid‐phase extraction cartridge (tC18 6cc Vac cartridge, 37‐ to 50‐μm particle size, 500 mg sorbent per cartridge; Waters)
  • 10‐ml syringe
  • 1.5‐ml microcentrifuge tubes
  • Vacuum concentrator (SpeedVac)
  • Rotator
  • Gel‐loading pipet tips (e.g., Starlab, cat. no. I1022‐0600)
  • Tabletop centrifuge
  • Solid‐phase extraction (SPE) disks for StageTips:
  • Empore SPE Disks, Cation Exchange‐SR (SCX), 47‐mm diameter (Sigma‐Aldrich, cat. no. 66889‐U)
  • Empore SPE Disks, C18, 47‐mm diameter (Sigma‐Aldrich, cat. no. 66883‐U)
  • 200‐μl pipet tips
  • StageTip adapters for microcentrifuge tubes (Sonation)
  • Instrumentation for online LC‐MS analysis (see Strategic Planning):
  • Orbitrap Q‐Exactive with nanoelectrospray source (Thermo Fisher Scientific)
  • Nanoscale UHPLC EASY‐nLC1000 system (Thermo Fisher Scientific)
  • Nanospray column for online UHPLC‐MS/MS (in‐house or New Objective)
  • Nanospray column heater (Sonation)
  • Additional reagents and equipment for determining protein concentration by Bradford protein assay (unit 3.4) and spectrophotometry (unit 3.1)
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Literature Cited

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