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.
NHEERL Proteomics Research Core, U.S. Environmental Protection Agency, Research Triangle Park, NC, USA.
Witold M
WM
Winnik
Robert M
RM
Dekroon
Joseph S Y
JS
Jeong
Mihaela
M
Mocanu
Jennifer B
JB
Robinette
Cristina
C
Osorio
Nedyalka N
NN
Dicheva
Eric
E
Hamlett
Oscar
O
Alzate
D006801:Humans; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism; D057825:Two-Dimensional Difference Gel Electrophoresis / Q000379:methods*
D010446:Peptide Fragments; D011506:Proteins; D014357:Trypsin