Skip to main content

Advertisement

SpringerLink
Log in

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

  • Protocol
  • Published:

From Nature Protocols

View current issue Submit your manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

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.

Similar content being viewed by others

References

  1. Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).

    Article  CAS  Google Scholar 

  2. Mann, M. & Jensen, O.N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 21, 255–261 (2003).

    Article  CAS  Google Scholar 

  3. Lill, J. Proteomic tools for quantitation by mass spectrometry. Mass Spectrom. Rev. 22, 182–194 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Lilley, K.S. & Friedman, D.B. All about DIGE: quantification technology for differential-display 2D-gel proteomics. Expert Rev. Proteomics 4, 401–409 (2004).

    Article  Google Scholar 

  5. Kolkman, A., Dirksen, E.H., Slijper, M. & Heck, A.J. Double standards in quantitative proteomics—direct comparative assessment of difference in gel electrophoresis and metabolic stable isotope labeling. Mol. Cell. Proteomics 3, 255–266 (2005).

    Article  Google Scholar 

  6. Gygi, S.O. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Ji, J. et al. Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. J. Chromatogr. A 745, 197–210 (2000).

    Article  CAS  Google Scholar 

  9. Ross, P.L., Huang, Y.L., Marchese, J.N. & Pappin, D.J. Multiplexed protein quantification in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154–1169 (2004).

    Article  CAS  Google Scholar 

  10. Mirgorodskaya, O.A. et al. Quantification of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using 18O-labeled internal standards. Rapid Comm. Mass Spectrom. 14, 1226–1232 (2000).

    Article  CAS  Google Scholar 

  11. Schmidt, A., Kellermann, J. & Lottspeich, F. A novel strategy for quantitative proteomics using isotope-coded protein labels. Proteomics 5, 4–15 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Asara, J.M. et al. In-gel stable isotope labeling (ISIL): a strategy for mass spectrometry-based relative quantification. J. Proteome Res. 5, 155–163 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Chakraborty, A. & Regnier, F.E. Global internal standard technology for comparative proteomics. J. Chromatogr. A 949, 173–184 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Zhang, X. et al. An automated method for the analysis of stable isotope labeling data in proteomics. J. Am. Soc. Mass Spectrom. 16, 1181–1191 (2005).

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, 02115, Massachusetts, USA

    John M Asara, Bin Zheng, Lisa A Maroney, Ning Wu & Lewis C Cantley

  2. Department of Pathology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    John M Asara

  3. Bindley Bioscience Center, Purdue University, West Lafayette, 47907, Indiana, USA

    Xiang Zhang

  4. Department of Systems Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Bin Zheng, Heather R Christofk, Ning Wu & Lewis C Cantley

Authors
  1. John M Asara
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bin Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lisa A Maroney
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Heather R Christofk
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ning Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lewis C Cantley
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John M Asara.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.7

  • Springer Nature Limited

Search

Navigation