Biology Reference
In-Depth Information
Chapter 5
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, and Oscar Alzate
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 identifi cation of DIGE-analyzed
proteins are discussed. Two examples are given: fi rst, a specifi c protein is used to test the sensitivity of the
2D DIGE/MALDI MS combination for protein quantifi cation and identifi cation, and second, several
proteins with and without the labels typically used in DIGE are identifi ed to demonstrate that these labels
do not alter MS-based protein identifi cation. Technical variations of protein gel spot preparation, in-gel
digestion, and mass spectral protein identifi cation are discussed.
Key words:
MALDI-TOF/TOF mass spectrometry, 2D DIGE, Gel spot picking, Destaining,
Dehydration, Enzymatic digestion, Peptide extraction, Data analysis, Proteomics
1. Introduction
Sensitivity of the 2D DIGE-MS (two-dimensional difference gel
electrophoresis mass spectrometry) (
1
) technique is vital given the
limited amount of tissue available for most proteomics experiments.
Hence a proof-of-principle study was undertaken to determine the
ability of 2D DIGE-MS to detect a protein standard at physiologi-
cal levels. The sensitivity of the approach was tested by detecting
decreasing amounts of the brain-derived neurotrophic factor
(BDNF) protein at the low picomole and subpicomole range. Also,
it is important to demonstrate that the Cy dyes, which are usually
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