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Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards

Nature Biotechnology volume 23, pages 617–621 (2005)Cite this article

Abstract

An important challenge for proteomics is to be able to compare absolute protein levels across biological samples1,2. Here we introduce an approach based on the use of culture-derived isotope tags (CDITs) for quantitative tissue proteome analysis. We cultured Neuro2A cells in a stable isotope-enriched medium and mixed them with mouse brain samples to serve as internal standards. Using CDITs, we identified and quantified a total of 1,000 proteins, 97–98% of which were expressed in both mouse whole brain and Neuro2A cells. CDITs also allow comprehensive and absolute protein quantification. Synthetic unlabeled peptides were used to quantify the corresponding proteins labeled with stable isotopes in Neuro2A cells, and the results were used to obtain the absolute amounts of 103 proteins in mouse whole brain. The expression levels correlated well with those in Neuro2A cells. Thus, the use of CDITs allows both relative and absolute quantitative proteome studies.

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Figure 1: Strategy of quantitative mouse brain proteomics using CDITs.
Figure 2: Validation data of CDIT strategy.
Figure 3: Total scheme for absolute quantification using CDITs.

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References

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

    Article  CAS  PubMed  Google Scholar 

  2. Sechi, S. & Oda, Y. Quantitative proteomics using mass spectrometry. Curr. Opin. Chem. Biol. 7, 70–77 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Oda, Y. et al. Quantitative chemical proteomics for identifying candidate drug targets. Anal. Chem. 75, 2159–2165 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Ranish, J.A. et al. The study of macromolecular complexes by quantitative proteomics. Nat. Genet. 33, 349–355 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Blagoev, B. et al. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat. Biotechnol. 21, 315–318 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Oda, Y., Huang, K., Cross, F.R., Cowburn, D. & Chait, B.T. Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. USA 96, 6591–6596 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Pasa-Tolic, L. et al. High-thoughput proteome-wide precision measurements of protein expression using mass spectrometry. J. Am. Chem. Soc. 121, 7949–7950 (1999).

    Article  CAS  Google Scholar 

  8. Wu, C.C., MacCoss, M.J., Howell, K.E., Matthews, D.E. & Yates, J.R. 3rd. Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis. Anal. Chem. 76, 4951–4959 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Jiang, H. & English, A.M. Quantitative analysis of the yeast proteome by incorporation of isotopically labeled leucine. J. Proteome Res. 1, 345–350 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. 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 

  11. Zhu, H., Pan, S., Gu, S., Bradbury, E.M. & Chen, X. Amino acid residue specific stable isotope labeling for quantitative proteomics. Rapid Mass Commun. Mass Spectrom. 16, 2115–2123 (2002).

    Article  CAS  Google Scholar 

  12. Sagane, K., Yamazaki, K., Mizui, Y. & Tanaka, I. Cloning and chromosomal mapping of mouse ADAM11, ADAM22 and ADAM23. Gene 236, 79–86 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. MacCoss, M.J., Wu, C.C., Liu, H., Sadygov, R. & Yates, J.R. 3rd. A correlation algorithm for the automated quantitative analysis of shotgun proteomics data. Anal. Chem. 75, 6912–6921 (2003).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  15. Sakai, J., Kojima, S., Yanagi, K. & Kanaoka, M. (18)O-labeling quantitative proteomics using an ion trap mass spectrometer. Proteomics 5, 16–23 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Parker, K.C. et al. Depth of proteome issues: a yeast isotope-coded affinity tag reagent study. Mol. Cell. Proteomics 3, 625–659 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. Mano, N., Oda, Y., Yamada, K., Asakawa, N. & Katayama, K. Simultaneous quantitative determination method for sphingolipid metabolites by liquid chromatography/ionspray ionization tandem mass spectrometry. Anal. Biochem. 244, 291–300 (1997).

    Article  CAS  PubMed  Google Scholar 

  18. Lensmeyer, G.L. & Poquette, M.A. Therapeutic monitoring of tacrolimus concentrations in blood: semi-automated extraction and liquid chromatography-electrospray ionization mass spectrometry. Ther. Drug. Monit. 23, 239–249 (2001).

    Article  CAS  PubMed  Google Scholar 

  19. Gunawan, S., Griswold, M.P. & Kahn, D.G. Liquid chromatographic-tandem mass spectrometric determination of amprenavir (agenerase) in serum/plasma of human immunodeficiency virus type-1 infected patients receiving combination antiretroviral therapy. J. Chromatogr. A. 914, 1–4 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Cass, R.T., Villa, J.S., Karr, D.E. & Schmidt, D.E. Jr. Rapid bioanalysis of vancomycin in serum and urine by high-performance liquid chromatography tandem mass spectrometry using on-line sample extraction and parallel analytical columns. Rapid Commun. Mass Spectrom. 15, 406–412 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Wilkinson, A.P., Wahala, K. & Williamson, G. Identification and quantification of polyphenol phytoestrogens in foods and human biological fluids. J. Chromatogr. B 777, 93–109 (2002).

    Article  CAS  Google Scholar 

  22. Collingridge, G.L. & Isaac, J.T. Functional roles of protein interactions with AMPA and kainate receptors. Neurosci. Res. 47, 3–15 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Lerma, J. Roles and rules of kainate receptors in synaptic transmission. Nat. Rev. Neurosci. 4, 481–495 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Barr, J.R. et al. Isotope dilution—mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I. Clin. Chem. 42, 1676–1682 (1996).

    CAS  PubMed  Google Scholar 

  25. Gerber, S.A., Rush, J., Stemman, O., Kirschner, M.W. & Gygi, S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. USA 100, 6940–6945 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Havlis, J. & Shevchenko, A. Absolute quantification of proteins in solutions and in polyacrylamide gels by mass spectrometry. Anal. Chem. 76, 3029–3036 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Katayama, H. et al. Efficient in-gel digestion procedure using 5-cyclohexyl-1-pentyl-beta-D-maltoside as an additive for gel-based membrane proteomics. Rapid Commun. Mass Spectrom. 18, 2388–2394 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Ishihama, Y., Rappsilber, J., Andersen, J.S. & Mann, M. Microcolumns with self-assembled particle frits for proteomics. J. Chromatogr. A 979, 233–239 (2002).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by funds from New Energy and Industrial Technology Development Organization, Japan (NEDO).

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Author notes

  1. Yasushi Ishihama and Toshitaka Sato: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Seeds Finding Technology, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, 300-2635, Ibaraki, Japan

    Yasushi Ishihama, Toshitaka Sato, Tsuyoshi Tabata, Norimasa Miyamoto, Koji Sagane, Takeshi Nagasu & Yoshiya Oda

Authors
  1. Yasushi Ishihama
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  2. Toshitaka Sato
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  3. Tsuyoshi Tabata
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  4. Norimasa Miyamoto
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  7. Yoshiya Oda
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Correspondence to Yoshiya Oda.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Reproducibility of quantitative procedures using CDIT (including from affinity purification to MS analysis) (PDF 62 kb)

Supplementary Table 2

Linearity and its reproducibility of quantitative procedures using CDIT (PDF 65 kb)

Supplementary Table 3

Dependence of MS signal ratios (heavy/light) on the mixing ratios (brain and Neuro2A) (PDF 16 kb)

Supplementary Table 4

Protein list quantified by CDIT and ICAT (PDF 112 kb)

Supplementary Table 5

Same sequence pair versus different sequence pair as internal standard (PDF 18 kb)

Supplementary Table 6

Number of quantified proteins in mouse hippocampus (PDF 92 kb)

Supplementary Table 7

Hippocampal proteins showing more than 2-fold change between kainate-treated and untreated mice (PDF 21 kb)

Supplementary Table 8

Absolute amount of proteins in Neuro2A cells and mouse brains (PDF 16 kb)

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Ishihama, Y., Sato, T., Tabata, T. et al. Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards. Nat Biotechnol 23, 617–621 (2005). https://doi.org/10.1038/nbt1086

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