Analysis of Proteins Using DIGE and MALDI Mass Spectrometry
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 wordsMALDI-TOF/TOF mass spectrometry 2D DIGE Gel spot picking Destaining Dehydration Enzymatic digestion Peptide extraction Data analysis Proteomics
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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