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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Abstract

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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Materials

Basic Protocol 1: Proteomics Analysis of Protein Deamidation of Single Proteins or Simple Protein Mixtures

  Materials
  • 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

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
  Cournoyer, J.J., Pittman, J.L., Ivleva, V.B., Fallows, E., Waskell, L., Costello, C.E., and O'Connor, P.B. 2005. Deamidation: Differentiation of aspartyl from isoaspartyl products in peptides by electron capture dissociation. Protein Sci. 14:452‐463.
  Cox, J. and Mann, M. 2008. MaxQuant enables high peptide identification rates, individualized p.p.b.‐range mass accuracies and proteome‐wide protein quantification. Nat. Biotechnol. 26:1367‐1372.
  Cox, J., Neuhauser, N., Michalski, A., Scheltema, R.A., Olsen, J.V., and Mann, M. 2011. Andromeda: A peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10:1794‐1805.
  Fang, X., Balgley, B.M., Wang, W., Park, D.M., and Lee, C.S. 2009. Comparison of multidimensional shotgun technologies targeting tissue proteomics. Electrophoresis 30:4063‐4070.
  Geiger, T. and Clarke, S. 1987. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide‐linked reactions that contribute to protein degradation. J. Biol. Chem. 262:785‐794.
  Hao, P., Guo, T., Li, X., Adav, S.S., Yang, J., Wei, M., and Sze, S.K. 2010. Novel application of electrostatic repulsion‐hydrophilic interaction chromatography (ERLIC) in shotgun proteomics: Comprehensive profiling of rat kidney proteome. J. Proteome Res. 9:3520‐3526.
  Hao, P., Ren, Y., Alpert, A.J., and Sze, S.K. 2011. Detection, evaluation and minimization of nonenzymatic deamidation in proteomic sample preparation. Mol. Cell Proteomics 10:O111.009381.
  Hao, P., Qian, J., Dutta, B., Cheow, E.S., Sim, K.H., Meng, W., Adav, S.S., Alpert, A., and Sze, S.K. 2012. Enhanced separation and characterization of deamidated peptides with RP‐ERLIC‐based multidimensional chromatography coupled with tandem mass spectrometry. J. Proteome Res. 11:1804‐1811.
  Hao, P., Ren, Y., Tam, J.P., and Sze, S.K. 2013. Correction of errors in tandem mass spectrum extraction enhances phosphopeptide identification. J. Proteome Res. 12:5548‐5557.
  Huang, L., Lu, J., Wroblewski, V.J., Beals, J.M., and Riggin, R.M. 2005. In vivo deamidation characterization of monoclonal antibody by LC/MS/MS. Anal. Chem. 77:1432‐1439.
  Krokhin, O.V., Antonovici, M., Ens, W., Wilkins, J.A., and Standing, K.G. 2006. Deamidation of ‐Asn‐Gly‐ sequences during sample preparation for proteomics: Consequences for MALDI and HPLC‐MALDI analysis. Anal. Chem. 78:6645‐6650.
  Li, X., Lin, C., and O'Connor, P.B. 2010. Glutamine deamidation: Differentiation of glutamic acid and gamma‐glutamic acid in peptides by electron capture dissociation. Anal. Chem. 82:3606‐3615.
  Ren, D., Pipes, G.D., Liu, D., Shih, L.Y., Nichols, A.C., Treuheit, M.J., Brems, D.N., and Bondarenko, P.V. 2009. An improved trypsin digestion method minimizes digestion‐induced modifications on proteins. Anal. Biochem. 392:12‐21.
  Robinson, N.E. 2002. Protein deamidation. Proc. Natl. Acad. Sci. U.S.A. 99:5283‐5288.
  Robinson, N.E., Lampi, K.J., McIver, R.T., Williams, R.H., Muster, W.C., Kruppa, G., and Robinson, A.B. 2005. Quantitative measurement of deamidation in lens betaB2‐crystallin and peptides by direct electrospray injection and fragmentation in a Fourier transform mass spectrometer. Mol. Vis. 11:1211‐1219.
  Roher, A.E., Lowenson, J.D., Clarke, S., Wolkow, C., Wang, R., Cotter, R.J., Reardon, I.M., Zurcher‐Neely, H.A., Heinrikson, R.L., Ball, M.J., and Greenberg, B.D. 1993. Structural alterations in the peptide backbone of beta‐amyloid core protein may account for its deposition and stability in Alzheimer's disease. J. Biol. Chem. 268:3072‐3083.
  Shimizu, T., Fukuda, H., Murayama, S., Izumiyama, N., and Shirasawa, T. 2002. Isoaspartate formation at position 23 of amyloid beta peptide enhanced fibril formation and deposited onto senile plaques and vascular amyloids in Alzheimer's disease. J. Neurosci. Res. 70:451‐461.
  Solstad, T. and Flatmark, T. 2000. Microheterogeneity of recombinant human phenylalanine hydroxylase as a result of nonenzymatic deamidations of labile amide containing amino acids. Effects on catalytic and stability properties. Eur. J. Biochem. 267:6302‐6310.
  Stroop, S.D. 2007. A modified peptide mapping strategy for quantifying site‐specific deamidation by electrospray time‐of‐flight mass spectrometry. Rapid Comm. Mass Spectrom. 21:830‐836.
  Takata, T., Oxford, J.T., Demeler, B., and Lampi, K.J. 2008. Deamidation destabilizes and triggers aggregation of a lens protein, betaA3‐crystallin. Protein Sci. 17:1565‐1575.
  Wilmarth, P.A., Tanner, S., Dasari, S., Nagalla, S.R., Riviere, M.A., Bafna, V., Pevzner, P.A., and David, L.L. 2006. Age‐related changes in human crystallins determined from comparative analysis of post‐translational modifications in young and aged lens: Does deamidation contribute to crystallin insolubility? J. Proteome Res. 5:2554‐2566.
  Yang, H., Fung, E.Y., Zubarev, A.R., and Zubarev, R.A. 2009. Toward proteome‐scale identification and quantification of isoaspartyl residues in biological samples. J. Proteome Res. 8:4615‐4621.
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