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In-gel stable isotope labeling for relative quantification using mass spectrometry

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.

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Figure 1: Overview of in-gel stable isotope labeling (ISIL).
Figure 2: ISIL-compatible polyacrylamide gels.
Figure 3: Changes in phosphorylation level using ISIL.
Figure 4: Low-abundance proteins stained with silver.

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Acknowledgements

The authors thank Takeda Chemical Industries for providing startup funds for the BIDMC Mass Spectrometry and Proteomics Core and NIH Grants GM56203 and CA089021 for providing support for this work.

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Correspondence to John M Asara.

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Asara, J., Zhang, X., Zheng, B. et al. In-gel stable isotope labeling for relative quantification using mass spectrometry. Nat Protoc 1, 46–51 (2006). https://doi.org/10.1038/nprot.2006.7

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