Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry

Nature Biotechnology volume 19, pages 946–951 (2001)Cite this article

Abstract

An approach to the systematic identification and quantification of the proteins contained in the microsomal fraction of cells is described. It consists of three steps: (1) preparation of microsomal fractions from cells or tissues representing different states; (2) covalent tagging of the proteins with isotope-coded affinity tag (ICAT) reagents followed by proteolysis of the combined labeled protein samples; and (3) isolation, identification, and quantification of the tagged peptides by multidimensional chromatography, automated tandem mass spectrometry, and computational analysis of the obtained data. The method was used to identify and determine the ratios of abundance of each of 491 proteins contained in the microsomal fractions of naïve and in vitro– differentiated human myeloid leukemia (HL-60) cells. The method and the new software tools to support it are well suited to the large-scale, quantitative analysis of membrane proteins and other classes of proteins that have been refractory to standard proteomics technology.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic representation of the quantitative proteomics procedure.
Figure 2: Multidimensional liquid chromatography tandem mass spectrometric analysis of a complex peptide mixture.
Figure 3: Post-MS processing of data from selected peptides.
Figure 4: Consistency of redundant protein quantification.
Figure 5: Categories of proteins identified in this study.

Similar content being viewed by others

References

  1. Oseroff, A.R., Robbins, P.W. & Burger, M.M. Cell surface membrane: biochemical aspects and biophysical probes. Annual Rev. Biochem. 42, 647–682 (1973).

    Article  CAS  Google Scholar 

  2. Liu, E. et al. The HER2 (c-erbB-2) oncogene is frequently amplified in in situ carcinomas of the breast. Oncogene 7, 1027–1032 (1992).

    CAS  PubMed  Google Scholar 

  3. Shak, S. Overview of the trastuzumab (Herceptin) anti-HER2 monoclonal antibody clinical program in HER2-overexpressing metastatic breast cancer. Herceptin Multinational Investigator Study Group. Semin. Oncol. 26, 71–77 (1999).

    CAS  PubMed  Google Scholar 

  4. Eccles, S.A. Monoclonal antibodies targeting cancer: “magic bullets” or just the trigger? Breast Cancer Res. 3, 86–90 (2001).

    Article  CAS  Google Scholar 

  5. Drews, J. Research and development. Basic science and pharmaceutical innovation. Nat. Biotechnol. 17, 406–408 (1999).

    Article  CAS  Google Scholar 

  6. Aebersold, R. & Goodlett, D.R. Mass spectrometry in proteomics. Chem. Rev. 101, 269–295 (2001).

    Article  CAS  Google Scholar 

  7. Molloy, M.P. et al. Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19, 837–844 (1998).

    Article  CAS  Google Scholar 

  8. Molloy, M.P. et al. Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 267, 2871–2881 (2000).

    Article  CAS  Google Scholar 

  9. Santoni, V., Molloy, M. & Rabilloud, T. Membrane proteins and proteomics: un amour impossible? Electrophoresis 21, 1054–1070 (2000).

    Article  CAS  Google Scholar 

  10. Rabilloud, T. et al. Analysis of membrane proteins by two-dimensional electrophoresis: comparison of the proteins extracted from normal or Plasmodium falciparum-infected erythrocyte ghosts. Electrophoresis 20, 3603–3610 (1999).

    Article  CAS  Google Scholar 

  11. Santoni, V. et al. Large scale characterization of plant plasma membrane proteins. Biochimie 81, 655–661 (1999).

    Article  CAS  Google Scholar 

  12. Simpson, R.J. et al. Proteomic analysis of the human colon carcinoma cell line (LIM 1215): development of a membrane protein database. Electrophoresis 21, 1707–1732 (2000).

    Article  CAS  Google Scholar 

  13. Birnie, G.D. The HL60 cell line: a model system for studying human myeloid cell differentiation. Br. J. Cancer Suppl. 9, 41–45 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Walter, P. & Blobel, G. Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol. 96, 84–93 (1983).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Eng, J., McCormack, A.L. & Yates, J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass. Spectrom. 5, 976–989 (1994).

    Article  CAS  Google Scholar 

  17. Link, A.J. et al. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682 (1999).

    Article  CAS  Google Scholar 

  18. Zhou, H., Watts, J.D. & Aebersold, R. A systematic approach to the analysis of protein phosphorylation. Nat. Biotechnol. 19, 375–378 (2001).

    Article  CAS  Google Scholar 

  19. Stamellos, K.D. et al. Subcellular localization of squalene synthase in rat hepatic cells. Biochemical and immunochemical evidence. J. Biol. Chem. 268, 12825–12836 (1993).

    CAS  PubMed  Google Scholar 

  20. Szkopinska, A., Swiezewska, E. & Karst, F. The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 267, 473–477 (2000).

    Article  CAS  Google Scholar 

  21. Goldstein, J.L. & Brown, M.S. Regulation of the mevalonate pathway. Nature 343, 425–430 (1990).

    Article  CAS  Google Scholar 

  22. Tansey, T.R. & Shechter, I. Squalene synthase: structure and regulation. Prog. Nucleic Acid Res. Mol. Biol. 65, 157–195 (2000).

    Article  Google Scholar 

  23. Gu, P., Ishii, Y., Spencer, T.A. & Shechter, I. Function–structure studies and identification of three enzyme domains involved in the catalytic activity in rat hepatic squalene synthase. J. Biol. Chem. 273, 12515–12525 (1998).

    Article  CAS  Google Scholar 

  24. Chen, H.T., Mehan, R.S., Gupta, S.D., Goldberg, I. & Shechter, I. Involvement of farnesyl protein transferase (FPTase) in Fc epsilonRI-induced activation of RBL-2H3 mast cells. Arch. Biochem. Biophys. 364, 203–208 (1999).

    Article  CAS  Google Scholar 

  25. Yokoyama, K., Goodwin, G.W., Ghomashchi, F., Glomset, J. & Gelb, M.H. Protein prenyltransferases. Biochem. Soc. Trans. 20, 489–494 (1992).

    Article  CAS  Google Scholar 

  26. Shechter, I. et al. Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase. J. Biol. Chem. 267, 8628–8635 (1992).

    CAS  PubMed  Google Scholar 

  27. Memon R.A. et al. Endotoxin, tumor necrosis factor, and interleukin-1 decrease hepatic squalene synthase activity, protein, and mRNA levels in Syrian hamsters. J. Lipid Res. 38, 1620–1691 (1997).

    CAS  PubMed  Google Scholar 

  28. Kiss, Z., Deli, E. & Kuo, J.F. Temporal changes in intracellular distribution of protein kinase C during differentiation of human leukemia KL60 cells induced by phorbol ester. FEBS Lett. 231, 41–46 (1988).

    Article  CAS  Google Scholar 

  29. Seibenhener, M.L. & Wooten, M.W. Heterogeneity of protein kinase C isoform expression in chemically induced HL60 cells. Exp. Cell. Res. 207, 183–188 (1993).

    Article  CAS  Google Scholar 

  30. Wooten, M.W., Seibenhener, M.L. & Soh, Y. Expression of protein kinase C isoforms in HL60 and phorbol ester resistant HL525 cells. Cytobios. 76, 19–29 (1993).

    CAS  PubMed  Google Scholar 

  31. Ohguchi, K., Banno, Y., Nakashima, S. & Nozawa, Y. Activation of membrane-bound phospholipase D by protein kinase C in HL60 cells: synergistic action of a small GTP-binding protein RhoA. Biochem. Biophys. Res. Commun. 211, 306–311 (1995).

    Article  CAS  Google Scholar 

  32. Aebersold, R., Hood, L.E. & Watts, J.D. Equipping scientists for the new biology. Nat. Biotechnol. 8, 359 (2000).

  33. Diehn, M., Eisen, M.B., Botstein, D. & Brown, P.O. Large-scale identification of secreted and membrane-associated gene products using DNA microarrays. Nat. Genet. 25, 58–62 (2000).

    Article  CAS  Google Scholar 

  34. Gygi, S.P., Corthals, G.L., Zhang, Y., Rochon, Y. & Aebersold, R. Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc. Natl. Acad. Sci. USA 97, 9390–9395 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank John Glomset, Julian Watts, Steve Gygi, and Beate Rist for helpful discussion and Julian Watts for critical reading of the manuscript. The work was supported by a grant from the University of Washington Royalty Research Fund, a grant from the Merck Genome Research Institute, grant no. 1R33CA84698 from the National Cancer Institute, and grant no. HL67569 from the National Institutes of Health.

Author information

Authors and Affiliations

  1. University of Connecticut Health Center, 263 Farmington Ave., Farmington, 06030-0002, CT

    David K. Han

  2. Institute for Systems Biology, 4225 Roosevelt Way NE, Seattle, 98105-6099, WA

    Jimmy Eng, Huilin Zhou & Ruedi Aebersold

Authors
  1. David K. Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jimmy Eng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huilin Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ruedi Aebersold
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ruedi Aebersold.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Han, D., Eng, J., Zhou, H. et al. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat Biotechnol 19, 946–951 (2001). https://doi.org/10.1038/nbt1001-946

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1001-946

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing