In-Gel Stable-Isotope Labeling (ISIL): a strategy for mass spectrometry-based relative quantification.
Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification.
Asara
John M
JM
Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115, USA. jasara@bidmc.harvard.edu
Zhang
Xiang
X
Zheng
Bin
B
Christofk
Heather H
HH
Wu
Ning
N
Cantley
Lewis C
LC
D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis*; D010748:Phosphopeptides; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*
D010455:Peptides; D010748:Phosphopeptides