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Analysis of proteins using DIGE and MALDI mass spectrometry
Citation:
WINNIK, W. M., R. DeKroon, M. Mocanu, E. Hamlett, C. Osorio, J. Robinette, N. Dicheva, AND O. Alzate. Analysis of proteins using DIGE and MALDI mass spectrometry. Chapter 854, John M. Walker (ed.), Methods in Molecular Biology. Humana Press Incorporated, Totowa, NJ, 2012(854):47-66, (2012).
Impact/Purpose:
Differential Gel Electrophoresis (DIGE) is a common technique for separation of proteins usually aimed at characterizing differential protein expression (l) in quantitative proteomics. Following protein separation and statistical analysis of differentially expressed spots, proteins are usually identified by a combination of enzymatic digestion followed by peptide analysis by mass spectrometry (2). In this chapter, methods for spot picking, enzymatic digestion and MALDI mass spectrometry for protein identification of DIGE-separated proteins are discussed. An example using a specific protein to test the sensitivity of the 2D-DIGE/MS combination to quantify and identify this protein is given. Technical variations of protein gel spot preparation, in-gel digestion, and mass spectral identification by MALDI are discussed.
Description:
In this work the sensitivity of the quantitative proteomics approach 2D-DIGE/MS (twoDimensional Difference Gel Electrophoresis / Mass Spectrometry) was tested by detecting decreasing amounts of a specific protein at the low picomole and sub-picomole range. Sensitivity of the 2D-DIGE / MS technique is vital given the limited amount of tissue available for most proteomics experiments. Hence this proof-of-principle study was undertaken to determine the ability of 2D-DIGE / MS to detect a protein standard at physiological levels. Several control experiments were carried out to demonstrate detection at the picomol and the sub-picomol (nanogram) range. Although mass spectrometry-based protein identification from 2D-DIGE gels is a well established technique, it has multiple procedural variations used in different laboratories. However, most of these technical variations share the following common steps: (a) spot picking from acrylamide gels, (b) protein destaining, (c) gel dehydration, (d) enzymatic digestion of the proteins and extraction of resulting peptides, (e) mass spectral analysis, and (f) software MS data analysis and verification of the results.