Protocol abstract
Nature Protocols 1, - 46 - 51 (2006)
Published online: 27 June 2006 | doi:10.1038/nprot.2006.7
Subject Category: Spectroscopy and structural analysis
In-gel stable isotope labeling for relative quantification using mass spectrometry
John M Asara1,2, Xiang Zhang3, Bin Zheng1,4, Lisa A Maroney1, Heather R Christofk4, Ning Wu1,4 & Lewis C Cantley1,4
Abstract
Although differences in protein staining intensity can often be visualized by difference gel electrophoresis, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. We present a protocol for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using in-gel stable isotope labeling. In this protocol protein extracts from any source treated under two experimental conditions are resolved in two separate lanes by gel electrophoresis. Parallel gel regions of interest are reacted separately with either light or heavy isotope-labeled reagents, and the gel slices are then combined and digested with proteases. The resulting peptides are then analyzed by liquid chromatography/mass spectrometry (LC/MS) to determine relative abundance of light- and heavy-isotope lysine-containing peptide pairs and analyzed by LC/MS/MS for identification of sequence and modifications. This protocol should take approximately 24–26 h to complete, including the incubation time for proteolytic digestion. Additional time will be needed for data analysis and interpretation.
- Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115, USA.
- Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
- Bindley Bioscience Center, Purdue University, West Lafayette, Indiana 47907, USA.
- Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Correspondence to: John M Asara1,2 e-mail: jasara@bidmc.harvard.edu
nature-products
A-Z product listing
- C18 resin (5
m, 100 Å) column packing material(Michrom Bioresources, Inc.) - D6-acetic anhydride(Sigma-Aldrich, Inc.)
- LTQ-FT(Thermo Electron Corp.)
- LTQ-Orbitrap(Thermo Electron Corp.)
- Microcapillary columns(New Objective, Inc.)
- Modified sequencing-grade trypsin(Promega Corp.)
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