Quantitative Protein Analysis Using Enzymatic [18O]Water Labeling

Mary Joan Castillo1, Kristy J. Reynolds2, Alexander Gomes1, Catherine Fenselau2, Xudong Yao1

1 Department of Chemistry, University of Connecticut, Storrs, Connecticut, 2 Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 23.4
DOI:  10.1002/0471140864.ps2304s76
Online Posting Date:  April, 2014
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Abstract

This unit describes the stepwise procedure for differential oxygen isotope labeling of peptides and mass spectrometric quantification of relative protein levels in comparative proteomic experiments. The [18O] labeling of peptides happens at the peptide C‐terminus and is achieved via the enzymatic oxygen exchange of tryptic peptides via catalysis of immobilized trypsin. Experimental considerations in effective incorporation and stabilization of the oxygen labels are discussed. Methods for mass spectrometric quantification of peptides with differential [16O] and [18O] isotopes are presented. Curr. Protoc. Protein Sci. 76:23.4.1‐23.4.9. © 2014 by John Wiley & Sons, Inc.

Keywords: 18O‐labeling; enzymatic oxygen labeling; quantitative proteomics; immobilized trypsin; back‐exchange

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparing a Protein Sample for Proteolytic Digestion and Enzymatic 18O Labeling
  • Support Protocol 1: Desalting Peptide Mixtures from Trypsin Digestion of Proteins or the Pooled Mixture of Solutions of Enzymatic Oxygen Exchange Reactions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparing a Protein Sample for Proteolytic Digestion and Enzymatic 18O Labeling

  Materials
  • Protein samples
  • 50 mM Tris·Cl, pH 8.0 ( )
  • Urea (Sigma)
  • 100 mM Dithiothreitol (DTT; Sigma)
  • 0.5 M iodoacetamide (Sigma)
  • 10 mM Tris·Cl, pH 7.4 ( )
  • Modified trypsin, sequencing grade (Promega)
  • 10% Trifluoroacetic acid (TFA; Pierce)
  • Immobilized trypsin on Poros materials (Applied Biosystems)
  • Deionized water (Direct‐Q3; Millipore or made from similar device)
  • H 218O, 97% (Cambridge Isotope Laboratories)
  • Acetonitrile, anhydrous
  • 0.2% Formic acid (FA; Sigma)
  • Benchtop rotator
  • BioSpin 6 column (BioRad)
  • pH paper
  • 37°C incubator
  • SpeedVac
  • Lyophilizer
  • Benchtop centrifuge
  • Microcentrifuge
  • IsoPro 3.0 software (http://members.aol.com/msmssoft)
  • Additional reagents and equipment for mass spectrometry (Chapter 16) and desalting the sample (Support Protocol)

Support Protocol 1: Desalting Peptide Mixtures from Trypsin Digestion of Proteins or the Pooled Mixture of Solutions of Enzymatic Oxygen Exchange Reactions

  Materials
  • Solution A: 20% formic acid (FA) in deionized (DI) H 2O
  • Peptide sample (see the Basic Protocol)
  • Solution C: [0.5% acetonitrile (ACN) + 0.1% TFA] in DI H 2O
  • Oasis HLB Material (Waters) packed in‐house in TopTips (Glygen)
  • Solution B: 70% ACN + 30% (0.2% FA in DI H 2O)
  • Solution D: (0.5% ACN + 0.2% FA) in DI H 2O
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Figures

Videos

Literature Cited

Literature Cited
   Bonenfant, D. , Schmelzle, T. , Jacinto, E. , Crespo, J.L. , Mini, T. , Hall, M.N. , and Jeno, P. 2003. Quantitation of changes in protein phosphorylation: A simple method based on stable isotope labeling and mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 100:880‐885.
   Fenselau, C. and Yao, X. 2009. 18O2‐labeling in quantitative proteomic strategies: A status report. J. Proteome Res. 8:2140‐2143.
   Jiang, H. , Ramos, A.A. , and Yao, X. 2010. Targeted quantitation of overexpressed and endogenous cystic fibrosis transmembrane conductance regulator using multiple reaction monitoring tandem mass spectrometry and oxygen stable isotope dilution. Anal. Chem. 82:336‐342.
   Reynolds, K.J. , Yao, X. , and Fenselau, C. 2002. Proteolytic 18O labeling for comparative proteomics: Evaluation of endoprotease Glu‐C as the catalytic agent. J. Proteome Res. 1:27‐33.
   Wang, S. , Bobst, C.E. , and Kaltashov, I.A. 2011. Pitfalls in protein quantitation using acid‐catalyzed O18 labeling: Hydrolysis‐driven deamidation. Anal. Chem. 83:7227‐7232.
   Yao, X. , Freas, A. , Ramirez, J. , Demirev, P.A. , and Fenselau, C. 2001. Proteolytic 18O labeling for comparative proteomics: Model studies with two serotypes of adenovirus. Anal. Chem. 73:2836‐2842.
   Yao, X. , Afonso, C. , and Fenselau, C. 2003. Dissection of proteolytic 18O labeling: Endoprotease‐catalyzed 16O‐to‐18O exchange of truncated peptide substrates. J. Proteome Res. 2:147‐152.
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