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Analysis of Proteins Using DIGE and MALDI Mass Spectrometry

  • Witold M. Winnik
  • Robert M. DeKroon
  • Joseph S. Y. Jeong
  • Mihaela Mocanu
  • Jennifer B. Robinette
  • Cristina Osorio
  • Nedyalka N. Dicheva
  • Eric Hamlett
  • Oscar AlzateEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 854)

Abstract

Difference gel electrophoresis (DIGE) is a common technique for characterizing differential protein expression in quantitative proteomics. Usually a combination of enzymatic digestion and peptide analysis by mass spectrometry is used to identify differentially expressed proteins following separation and statistical analysis by DIGE. In this chapter, methods for gel spot picking, enzymatic digestion, and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) for protein identification of DIGE-analyzed proteins are discussed. Two examples are given: first, a specific protein is used to test the sensitivity of the 2D DIGE/MALDI MS combination for protein quantification and identification, and second, several proteins with and without the labels typically used in DIGE are identified to demonstrate that these labels do not alter MS-based protein identification. Technical variations of protein gel spot preparation, in-gel digestion, and mass spectral protein identification are discussed.

Key words

MALDI-TOF/TOF mass spectrometry 2D DIGE Gel spot picking Destaining Dehydration Enzymatic digestion Peptide extraction Data analysis Proteomics 

Notes

Acknowledgments

The research described here was possible thanks to financial support from the Systems-Proteomics Center—University of North Carolina at Chapel Hill. We would like to thank Dr. Roger Madison (Duke University Medical Center) for helping with the acquisition and analysis of human BDNF.

Disclaimer: The research described in this article was reviewed by the National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Witold M. Winnik
    • 1
  • Robert M. DeKroon
    • 2
  • Joseph S. Y. Jeong
    • 2
  • Mihaela Mocanu
    • 3
  • Jennifer B. Robinette
    • 3
  • Cristina Osorio
    • 3
  • Nedyalka N. Dicheva
    • 4
  • Eric Hamlett
    • 2
  • Oscar Alzate
    • 2
    Email author
  1. 1.NHEERL Proteomics Research CoreU.S. Environmental Protection AgencyResearch Triangle ParkUSA
  2. 2.Department of Cell and Developmental BiologyUniversity of North CarolinaChapel HillUSA
  3. 3.UNC Systems-Proteomics Center, Program of Molecular Biology and Biotechnology, School of MedicineUniversity of North CarolinaChapel HillUSA
  4. 4.Program of Molecular Biology and Biotechnology, School of MedicineUniversity of North CarolinaChapel HillUSA

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