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In-gel stable isotope labeling for relative quantification using mass spectrometry
John M Asara1, Xiang Zhang, Bin Zheng
1Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115, USA. jasara@bidmc.harvard.edu
Nature Protocols
|April 5, 2007
Summary
This study introduces a new in-gel stable isotope labeling method for precise protein quantification and analysis of post-translational modifications. This technique overcomes limitations of traditional methods, enabling accurate measurement of specific protein changes.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- Difference gel electrophoresis is limited by abundant proteins obscuring less abundant ones.
- Quantifying post-translational modifications accurately remains challenging with current methods.
Purpose of the Study:
- To present a novel protocol for quantifying specific protein abundance changes.
- To enable accurate quantification of specific protein modifications using in-gel stable isotope labeling.
Main Methods:
- Protein extracts from two experimental conditions are separated by gel electrophoresis.
- Parallel gel regions are labeled with light or heavy isotopes, combined, and digested.
- Peptides are analyzed by liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS.
Main Results:
- The protocol allows for relative quantification of light- and heavy-isotope labeled peptide pairs.
- LC/MS/MS enables identification of peptide sequences and associated modifications.
- The method provides a robust way to analyze changes in protein abundance and modifications.
Conclusions:
- This in-gel stable isotope labeling protocol offers a sensitive and accurate approach for proteomic analysis.
- It effectively addresses limitations in quantifying specific proteins and their modifications.
- The protocol is applicable to various protein sources and experimental conditions.
