Proteomic Analysis of Protein Deamidation

Piliang Hao1, Siu Kwan Sze2

1 Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, 2 School of Biological Sciences, Nanyang Technological University, Singapore
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 24.5
DOI:  10.1002/0471140864.ps2405s78
Online Posting Date:  November, 2014
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Deamidation of asparagines and glutamines occurs spontaneously in proteins and results in protein degradation. Deamidation of asparaginyl residues in proteins produces a mixture of asparaginyl, n‐aspartyl, and isoaspartyl residues, which has been linked to the pathology of some neurodegenerative diseases. However, accurate proteomic analysis of deamidation is challenging since it occurs quickly during conventional proteomic sample preparation, and the co‐elution of the two resulting isomeric deamidated peptides in reversed‐phase liquid chromatography (RPLC) compromises their identification and quantification using RPLC‐MS/MS. To overcome these difficulties, a novel sample preparation protocol to minimize artificial deamidation has been developed alongside an offline RP‐ERLIC‐MS/MS (reversed‐phase chromatography fractionation followed by electrostatic repulsion‐hydrophilic interaction chromatography coupled with MS/MS) strategy to separate and quantify the three deamidation products from the same peptide on a proteome‐wide scale. These protocols are detailed in this unit. © 2014 by John Wiley & Sons, Inc.

Keywords: nonenzymatic deamidation; ERLIC; RP‐ERLIC‐MS/MS; mass spectrometry; artificial deamidation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Proteomics Analysis of Protein Deamidation of Single Proteins or Simple Protein Mixtures
  • Basic Protocol 2: Proteomics Analysis of Protein Deamidation in Complex Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Proteomics Analysis of Protein Deamidation of Single Proteins or Simple Protein Mixtures

  • Bovine serum albumin (BSA)
  • Chicken ovalbumin
  • Lysis buffer (see recipe)
  • 100 mM dithiotreitol (DTT) in water
  • 0.5 M iodoacetamide (IAA) in water
  • 50 mM ammonium acetate, pH 6
  • Sequencing‐grade trypsin solution: 1 mg/ml in 50 mM acetic acid
  • 10% trifluoroacetic acid (TFA)
  • Methanol
  • 70% acetonitrile/0.1% TFA
  • 85% acetonitrile, 0.1% formic acid (FA)
  • Capillary column (200 μm × 15–cm) packed with PolyWAX LP anion‐exchange bulk material (5 μm, 300 Å; PolyLC)
  • ERLIC‐MS/MS mobile phase A: 0.1% formic acid (FA) in acetonitrile
  • ERLIC‐MS/MS mobile phase B: 0.1% formic acid (FA) in water
  • 1.5‐ml tubes
  • Vortexer
  • Centrifuge
  • 37°C water bath
  • Sep‐Pak C18 cartridges (Waters)
  • Pipets
  • SpeedVac (Thermo Electron)
  • Q Exactive mass spectrometer (Thermo Fisher) coupled with a Dionex Ultimate 3000 RSLCnano system

Basic Protocol 2: Proteomics Analysis of Protein Deamidation in Complex Samples

  • Sprague‐Dawley rat livers (Centre for Animal Care, National University of Singapore)
  • 1× PBS
  • Liquid nitrogen
  • Lysis buffer (see recipe)
  • 2‐D Quant Kit (GE Healthcare)
  • 100 mM DTT
  • 0.5 M IAA
  • 50 mM ammonium acetate, pH 6
  • Sequencing‐grade trypsin solution: 1 mg/ml in 50 mM acetic acid
  • 10% TFA
  • Sep‐Pak C18 cartridges
  • Methanol
  • 70% acetonitrile/0.1% TFA
  • RPLC fractionation mobile phase A: 0.1% formic acid (FA) in water
  • BioBasic C18 column (4.6 × 250–mm, 5 μm, 300 Å, Thermo Scientific)
  • RPLC fractionation mobile phase B: 0.1% FA in acetonitrile
  • 85% acetonitrile/0.1% FA
  • Mortar and pestle
  • 2‐ml tubes
  • Vibra Cell high‐intensity ultrasonic processor (Jencons Scientific)
  • Refrigerated centrifuge
  • 37°C water bath
  • Pipets
  • SpeedVac
  • Shimadzu Prominence UFLC system
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Literature Cited

Literature Cited
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