Enrichment of Methylated Peptides Using an Antibody‐free Approach for Global Methylproteomics Analysis

Keyun Wang1, Mingliang Ye1

1 University of Chinese Academy of Sciences, Beijing
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.18
DOI:  10.1002/cpps.49
Online Posting Date:  February, 2018
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Protein methylation is receiving increasing attention for its important role in regulating diverse biological processes, including epigenetic regulation of gene transcription, RNA processing, DNA damage repair, and signal transduction. Proteome level analysis of protein methylation requires the enrichment of various forms of methylated peptides. Unfortunately, immunoaffinity purification can only enrich a subset of them due to the lack of pan‐specific antibodies. Chromatography‐based methods, however, can enrich methylated peptides in a global manner. Here we present a chromatography‐based approach for highly efficient enrichment of methylated peptides. Protocols for the of high pH SCXtip preparation and methyl‐peptide purification are described in detail. Key points such as cell culture in hM‐SILAC medium and protein digestion by multiple endopeptidases are also presented. This technique allows the simultaneous analysis of both lysine and arginine methylation and improved performance for methyl‐arginine identification. © 2018 by John Wiley & Sons, Inc.

Keywords: hM‐SILAC; mass spectrometry; post‐translational modification; protein methylation; proteomics

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Table of Contents

  • Introduction
  • Basic Protocol 1: Large‐Scale Analysis of Protein Methylation by High pH SCXtip Separation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Large‐Scale Analysis of Protein Methylation by High pH SCXtip Separation

  • Cells of interest
  • hM‐SILAC medium (see recipe)
  • Lysis buffer (see recipe)
  • Bradford reagent
  • Dithiothreitol (DTT)
  • Iodoacetamide
  • 50 mM ammonium bicarbonate (NH 4HCO 3)
  • Trypsin, MS grade (Hualishi Technologies, cat. no. HLS TRY001C)
  • Lysyl endopeptidase, MS grade (Wako Chemicals, cat. no. 129‐02541)
  • Calcium chloride anhydrous (CaCl 2)
  • 10× activation buffer (see recipe)
  • Clostripain (arg‐C, Worthington Biochemical, cat. no. CLS001643)
  • Trifluoroacetic acid (TFA)
  • Methanol
  • Acetonitrile (ACN)
  • Polar MC‐SP, 30 μm (SCX beads; Sepax Technologies, cat. no. 283030)
  • 5 mM BRUB buffer, pH 12 (see recipe)
  • SCXtip buffers (see recipe)
  • RP‐HPLC solvents (see recipe)
  • 10‐kDa MWCO centrifugal filters (Millipore Sigma, cat. no. UFC501096)
  • HLB extraction cartridge, 10 mg (Waters, cat. no. 186000383) and 60 mg (Waters, cat. no. WAT094226)
  • Degreasing cotton (Sinopharm Chemical Regent, cat. no. 92653866)
  • Pipet tips, 200 μl
  • Empore SDB‐XC extraction disks (3M, cat. no. 2240)
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Literature Cited

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