The Isotope‐Coded Affinity Tag Method for Quantitative Protein Profile Comparison and Relative Quantitation of Cysteine Redox Modifications

James Chun Yip Chan1, Lei Zhou2, Eric Chun Yong Chan1

1 Department of Pharmacy, National University of Singapore, 2 Singapore Eye Research Institute
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 23.2
DOI:  10.1002/0471140864.ps2302s82
Online Posting Date:  November, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The isotope‐coded affinity tag (ICAT) technique has been applied to measure pairwise changes in protein expression through differential stable isotopic labeling of proteins or peptides followed by identification and quantification using a mass spectrometer. Changes in protein expression are observed when the identical peptide from each of two biological conditions is identified and a difference is detected in the measurements comparing the peptide labeled with the heavy isotope to the one with a normal isotopic distribution. This approach allows the simultaneous comparison of the expression of many proteins between two different biological states (e.g., yeast grown on galactose versus glucose, or normal versus cancer cells). Due to the cysteine‐specificity of the ICAT reagents, the ICAT technique has also been applied to perform relative quantitation of cysteine redox modifications such as oxidation and nitrosylation. This unit describes both protein quantitation and profiling of cysteine redox modifications using the ICAT technique. © 2015 by John Wiley & Sons, Inc.

Keywords: isotope‐coded affinity tag (ICAT); quantitative protein profiling; tandem mass spectrometry; liquid chromatography; cysteine thiol modification; redox proteomics

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Labeling Proteins with Acid‐Cleavable Isotope‐Coded Affinity Tag Reagent
  • Basic Protocol 2: Purification and Fractionation of Peptides Labeled with Isotope‐Coded Affinity Tags by Strong Cation‐Exchange Chromatography
  • Basic Protocol 3: Affinity Purification with Avidin Affinity Cartridges of Peptides Labeled with Isotope‐Coded Affinity Tags
  • Basic Protocol 4: Cleavage of the Biotin Affinity Tag from Peptides Labeled with Isotope‐Coded Affinity Tags
  • Basic Protocol 5: Quenching of Thiol Redox State Through Trichloroacetic Acid Acidification
  • Basic Protocol 6: Duplex Labeling of Reduced and Modified Thiols with Light and Heavy ICAT Reagents
  • Basic Protocol 7: Monoplex Labeling of Modified Thiols with Light and Heavy ICAT Reagents
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Labeling Proteins with Acid‐Cleavable Isotope‐Coded Affinity Tag Reagent

  Materials
  • Protein pellets, dried, from control and test samples
  • Labeling buffer: 0.05% (w/v) SDS/50 mM Tris·Cl, pH 8.3/5 mM EDTA, pH 8.0/6 M urea; prepare fresh from stock solutions ( appendix 2E)
  • Bovine IgG
  • 250 mM Tris(2‐carboxyethyl) phosphine (TCEP)
  • 50 mM Tris·Cl, pH 8.3 ( appendix 2E)
  • Cleavable ICAT reagents (heavy and light; Applied Biosystems)
  • 1 M dithiothreitol (DTT; appendix 2E)
  • Sequencing‐grade trypsin (Promega)
  • 6 N acetic acid
  • Tube rocker (e.g., Baxter Scientific Products)
  • Additional reagents and equipment for determining protein concentration (unit 3.4), SDS–polyacrylamide gel electrophoresis (SDS‐PAGE; unit 10.1), and silver staining (unit 10.5)

Basic Protocol 2: Purification and Fractionation of Peptides Labeled with Isotope‐Coded Affinity Tags by Strong Cation‐Exchange Chromatography

  Materials
  • ICAT‐labeled protein sample (see protocol 1)
  • 3 N phosphoric acid
  • Cation‐exchange buffer B: 10 mM potassium phosphate/600 mM KCl/25% (v/v) acetonitrile, pH 3.0; store at room temperature
  • Cation‐exchange buffer A: 10 mM potassium phosphate/25% (v/v) acetonitrile, pH 3.0; store at room temperature
  • 2.1 × 200–mm PolySULFOETHYL A column (PolyLC)
  • High‐performance liquid chromatography (HPLC) system with binary pump and fraction collector
  • 1‐ml glass vials with polyethylene snap caps (e.g., Waters Chromatography)
  • 2‐ml microcentrifuge tubes (e.g., Micro Tube; Sarstedt)

Basic Protocol 3: Affinity Purification with Avidin Affinity Cartridges of Peptides Labeled with Isotope‐Coded Affinity Tags

  Materials
  • Loading buffer: 2× PBS ( appendix 2E)
  • Cation‐exchange fractions (see protocol 2)
  • Wash buffer 1: 1× PBS ( appendix 2E)
  • Wash buffer 2: 50 mM ammonium bicarbonate, pH 8.3/20% (v/v) methanol
  • Elution buffer: 0.4% (v/v) trifluoroacetic acid (TFA)/30% (v/v) acetonitrile
  • Avidin cartridges and cartridge holders (Applied Biosystems)
  • Ring stand for mounting cartridge
  • 1000‐μl gas‐tight syringe with stainless steel needle (e.g., Hamilton)
  • 1‐ml glass vials with polyethylene snap caps (e.g., Waters Chromatography)
NOTE: All buffers should be stored up to 3 months at 4°C.

Basic Protocol 4: Cleavage of the Biotin Affinity Tag from Peptides Labeled with Isotope‐Coded Affinity Tags

  Materials
  • Affinity‐purified ICAT‐labeled peptide fractions in glass vials (see protocol 3)
  • Cleaving reagents A and B (Applied Biosystems)
  • Appropriate solvent for microcapillary liquid chromatography–tandem mass spectrometry (μLC‐MS/MS) analysis
  • Speedvac evaporator (Savant)
  • 1000‐μl glass syringe with stainless steel needle (e.g., Hamilton)
  • 1‐ml glass vials with polyethylene snap caps (e.g., Waters Chromatography)

Basic Protocol 5: Quenching of Thiol Redox State Through Trichloroacetic Acid Acidification

  Materials
  • Plated mammalian cells cultured in 6‐well plates
  • Ice
  • Ice‐cold serum‐free culture medium
  • Ice‐cold 10% (w/v) trichloroacetic acid (TCA)
  • Degassed labeling buffer ( protocol 1)
  • Bicinchoninic acid (BCA) or Bradford protein assay
  • Cell scraper
  • 2‐ml microcentrifuge tubes
  • Microcentrifuge

Basic Protocol 6: Duplex Labeling of Reduced and Modified Thiols with Light and Heavy ICAT Reagents

  Additional Materials (also see protocol 1)
  • Acetonitrile
  • Solubilized protein sample (prepared in step 6 of protocol 5)
  • Acetone prechilled at −20°C
  • 50 mM Tris(2‐carboxyethyl) phosphine (TCEP) (available as reducing reagent; ABSciex)
  • Thermomixer (Eppendorf)
  • Vortex mixer
  • Centrifuge
  • Additional reagents and equipment for cation‐exchange and avidin purification of ICAT‐labeled peptides (Basic Protocols protocol 22 and protocol 33)

Basic Protocol 7: Monoplex Labeling of Modified Thiols with Light and Heavy ICAT Reagents

  Additional Materials (also see protocol 1)
  • 100 mM iodoacetamide (IAM) dissolved in degassed labeling buffer
  • Protein sample (prepared in step 6 of protocol 5)
  • Acetone prechilled at −20°C
  • 50 mM Tris(2‐carboxyethyl) phosphine (TCEP) (available as Reducing Reagent (ABSciex)
  • Thermomixer (Eppendorf)
  • Vortex mixer
  • Centrifuge
  • Additional reagents and equipment for cation‐exchange and avidin purification of ICAT‐labeled peptides (Basic Protocols protocol 22 and protocol 33)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Baliga, N.S., Pan, M., Goo, Y.A., Yi, E.C., Goodlett, D.R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W.V., and Hood, L. 2002. Coordinate regulation of energy transduction modules in Halobacterium sp. analyzed by a global systems approach. Proc. Natl. Acad. Sci. U.S.A. 99:4913–14918. doi: 10.1073/pnas.192558999.
  Flory, M.R., Griffin, T.J., Martin, D., and Aebersold, R. 2002. Advances in quantitative proteomics using stable isotope tags. Trends Biotech. 12:S23‐S29.
  Fu, C., Hu, J., Liu, T., Ago, T., Sadoshima, J., and Li, H. 2008. Quantitative analysis of redox‐sensitive proteome with DIGE and ICAT. J. Proteome Res. 7:3789‐3802. doi: 10.1021/pr800233r.
  Garcia‐Santamarina, S., Boronat, S., Espadas, G., Ayte, J., Molina, H., and Hidalgo, E. 2011. The oxidized thiol proteome in fission yeast–optimization of an ICAT‐based method to identify H2O2‐oxidized proteins. J. Proteomics 74:2476‐2486. doi: 10.1016/j.jprot.2011.05.030.
  Go, Y.M., Roede, J.R., Orr, M., Liang, Y., and Jones, D.P. 2014. Integrated redox proteomics and metabolomics of mitochondria to identify mechanisms of cd toxicity. Toxicol. Sci. 139:59‐73. doi: 10.1093/toxsci/kfu018.
  Gonzalez‐Galarza, F.F., Lawless, C., Hubbard, S.J., Fan, J., Bessant, C., Hermjakob, H., and Jones, A.R. 2012. A critical appraisal of techniques, software packages, and standards for quantitative proteomic analysis. OMICS 16:431‐442. doi: 10.1089/omi.2012.0022.
  Goodlett, D.R. and Aebersold, R. 2001. Mass spectrometry in proteomics. Chem. Rev. 101:269‐295. doi: 10.1021/cr990076h.
  Goodlett, D.R. and Yi, E.C. 2002. Proteomics without polyacrylamide: Qualitative and quantitative uses of tandem mass spectrometry in proteome analysis. Funct. Integr. Genomics 2:138‐153. doi: 10.1007/s10142‐001‐0041‐3.
  Guevel, L., Lavoie, J.R., Perez‐Iratxeta, C., Rouger, K., Dubreil, L., Feron, M., Talon, S., Brand, M., and Megeney, L.A. 2011. Quantitative proteomic analysis of dystrophic dog muscle. J. Proteome Res.10:2465‐2478. doi: 10.1021/pr2001385.
  Guina, T., Purvine, S.O., Yi, E.C., Eng, J., Goodlett, D.R., Aebersold, R., and Miller, S.I. 2003. Quantitative proteomic analysis indicates increased synthesis of a quinolone by Pseudomonas aeruginosa isolates from cystic fibrosis airways. Proc. Natl. Acad. Sci. U.S.A. 100:2771‐2776. doi: 10.1073/pnas.0435846100.
  Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M., and Aebersold, R. 1999. Quantitative analysis of complex protein mixtures using isotope coded affinity tags. Nat. Biotechnol. 17:994‐999. doi: 10.1038/13690
  Han, D.K., Eng, J., Zhou, H., and Aebersold, R. 2001. Quantitative profiling of differentiation‐induced microsomal proteins using isotope‐coded affinity tags and mass spectrometery. Nat. Biotechnol. 19:946‐951. doi: 10.1038/nbt1001‐946.
  Hu, Z., Wang, L., Xie, Z., Zhang, X., Feng, D., Wang, F., Zuo, B., Liu, Z., Chen, Z., Yang, F., and Liu, L. 2011. Quantitative proteomics analysis of parthenogenetically induced pluripotent stem cells. Protein Cell 2:631‐646. doi: 10.1007/s13238‐011‐1081‐7.
  Kang, U.B., Ahn, Y., Lee, J.W., Kim, Y.H., Kim, J., Yu, M.H., Noh, D.Y., and Lee, C. 2010. Differential profiling of breast cancer plasma proteome by isotope‐coded affinity tagging method reveals biotinidase as a breast cancer biomarker. BMC Cancer 10:114. doi: 10.1186/1471‐2407‐10‐114.
  Kumar, V., Kleffmann, T., Hampton, M.B., Cannell, M.B., and Winterbourn, C.C. 2013. Redox proteomics of thiol proteins in mouse heart during ischemia/reperfusion using ICAT reagents and mass spectrometry. Free Radic. Biol. Med. 58:109‐117. doi: 10.1016/j.freeradbiomed.2013.01.021
  Leichert, L.I., Gehrke, F., Gudiseva, H.V., Blackwell, T., Ilbert, M., Walker, A.K., Strahler, J.R., Andrews, P.C., and Jakob, U. 2008. Quantifying changes in the thiol redox proteome upon oxidative stress in vivo. Proc. Natl. Acad. Sci. U.S.A. 105:8197‐8202. doi: 10.1073/pnas.0707723105.
  Murray, C.I., Uhrigshardt, H., O'Meally, R.N., Cole, R.N., and Van Eyk, J.E. 2012. Identification and quantification of S‐nitrosylation by cysteine reactive tandem mass tag switch assay. Mol. Cell Proteomics 11:M111 013441. doi: 10.1074/mcp.M111.013441
  Ranish, J.A., Yi, E.C., Leslie, D.M., Purvine, S.O., Goodlett, D.R., Eng, J., and Aebersold, R. 2003. The study of macromolecular complexes by quantitative proteomics. Nat. Genet. 33:349‐355. doi: 10.1038/ng1101.
  Shiio, Y., Donohoe, S., Yi, E.C., Goodlett, D.R., Aebersold, R., and Eisenman, R.N. 2002. Quantitative proteomic analysis of Myc oncoprotein function. EMBO J. 21:5088‐5096. doi: 10.1093/emboj/cdf525
  Smolka, M.B., Zhou, H., Purkayastha, S., and Aebersold, R. 2001. Optimization of the isotope‐coded affinity tag‐labeling procedure for quantitative proteome analysis. Anal. Biochem. 297:25‐31. doi: 10.1006/abio.2001.5318.
  Yi, E.C., Mrelli, M., Lee, H., Purvine, S.O., Aebersold, R., Aitchison, J.D., and Goodlett, D.R. 2002. Approaching complete peroxisome characterization by gas‐phase fractionation. Electrophoresis 23:3205‐3216. doi: 10.1002/1522‐2683(200209)23:18%3c3205::AID‐ELPS3205%3e3.0.CO;2‐Y.
  Zhang, X., Huang, B., Zhou, X., and Chen, C. 2010. Quantitative proteomic analysis of S‐nitrosated proteins in diabetic mouse liver with ICAT switch method. Protein Cell 1:675‐687. doi: 10.1007/s13238‐010‐0087‐x.
Internet Resources
  http://www.proteomecenter.org/software.php
  Includes details of proteomics software tools from the Institute for Systems Biology.
  http://www.absciex.com/Documents/Downloads/Literature/4333373C.pdf
  Provides further information on ABSciex's ICAT reagents.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library