Skip to main content

In-depth analysis of the human CSF proteome using protein prefractionation

Abstract

The identification of disease markers in human body fluids requires an extensive and thorough analysis of its protein constituents. In the present study, we have extended our analysis of the human cerebrospinal fluid (CSF) proteome using protein prefractional followed by shotgun mass spectrometry. After the removal of abundant protein components from the mixture with the help of immunodepletion affinity chromatography, we used either anion exchange chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to further subfractionate the proteins present in CSFs. Each protein subfraction was enzyme digested and analyzed by tandem mass spectrometry and the resulting data evaluated using the Spectrum Mill software. Different subfractionation methods resulted in the identification of a grant total of 259 proteins in CSF from a patient with normal pressure hydrocephalus. The greatest number of protein, 240 in total, were identified after prefractionating the CSF proteins by immunodepletion and SDS-PAGE. Immuno-depletion combined with anion exchange fractionation resulted in 112 proteins and 74 proteins were found when only immunodepletion of the CSF samples was carried out. All methods used showed a significant increase in the number of identified proteins as compared with nondepleted and unfractionated CSF sample analysis, which yielded only 38 protein identifications. The present work establishes a platform for future studies aimed at a detailed comparative proteome analysis of CSFs from different groups of patients suffering from various psychiatric and neurological disorders.

References

  1. 1.

    Holsboer, F. (2001). Antidepressant drug discovery in the postgenomic era. World J. Biol. Psychiatry 2:165–177.

    PubMed  CAS  Article  Google Scholar 

  2. 2.

    Reiber, H. (2001). Dynamics of brain-derived proteins in cerebrospinal fluid. Clin. Chim. Acta 310:173–186.

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Anderson, N.L. and Anderson, N.G. (1998). Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis 19:1853–1861.

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Klose, J. (1975). Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26:231–243.

    PubMed  CAS  Google Scholar 

  5. 5.

    O'Farrel, P.H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007–4021.

    Google Scholar 

  6. 6.

    Link, A.J. (2002). Multidimensional peptide separations in proteomics. Trends Biotechnol. 20(Suppl. 12):S8-S13.

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Maccarrone, G., Milfay, D., Birg, I., Rosenhagen, M., Grimm, R., Bailey, J., et al. (2004). Mining the human CSF proteome by immunodepletion and shotgun mass spectrometry. Electrophoresis 25:2402–2412.

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Eng, J., McCormack, J.R., and Yates, J. (1994). An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5:976–984.

    Article  CAS  Google Scholar 

  9. 9.

    Spectrum Mill MS Proteomics Workbench-Comprehensive tools for MS proteomics. 2003. Available at: http://www.agilent.com.

  10. 10.

    Anderson, N.L., Polanski, M., Pieper, R., Gatlin, T., Tirumalai, R.S., Conrads, T.P., et al. (2004). The human plasma proteome. Mol. Cell. Proteomics 3:311–326.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Chan, K.C., Lucas, D.A., Hise, D., Schaefer, C.F., Xiao, Z., Janini, G.M., et al. (2004). Analysis of the human serum proteome. Clin. Proteomics 1:101–225.

    Article  Google Scholar 

  12. 12.

    Reiber, H., Otto, M., Trendelenburg, C., and Wormek, A. (2001). Reporting cerebrospinal fluid data: knowledge base and interpretation software. Clin. Chem. Lab. Med. 39:324–332.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Sickmann, A., Dormeyer, W., Wortelkamp, S., Woitalla, D., Kuhn, W., and Meyer, H.E. (2002). Towards a high resolution separation of human cerebrospinal fluid. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 771:167–196.

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Davidsson, P., Paulson, L., Hesse, C., Blennow, K., and Nilsson, C.L. (2001). Proteome studies of human cerebrospinal fluid and brain tissue using a preparative two-dimensional electrophoresis prior to mass spectrometry. Proteomics 1:444–452.

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Christoph W. Turck PhD.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Maccarrone, G., Birg, I., Malisch, E. et al. In-depth analysis of the human CSF proteome using protein prefractionation. Clin Proteom 1, 333–364 (2004). https://doi.org/10.1385/CP:1:3-4:333

Download citation

Key Words

  • Brain disease
  • cerebrospinal fluid
  • proteome
  • protein prefractionation