Advertisement

In-Depth Analysis of a Plasma or Serum Proteome Using a 4D Protein Profiling Method

  • Hsin-Yao Tang
  • Lynn A. Beer
  • David W. Speicher
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 728)

Abstract

Comprehensive proteomic analysis of human plasma or serum has been a major strategy used to identify biomarkers that serve as indicators of disease. However, such in-depth proteomic analyses are challenging due to the complexity and extremely large dynamic range of protein concentrations in plasma. Therefore, reduction in sample complexity through multidimensional pre-fractionation strategies is critical, particularly for the detection of low-abundance proteins that have the potential to be the most specific disease biomarkers. We describe here a 4D protein profiling method that we developed for comprehensive proteomic analyses of both plasma and serum. Our method consists of abundant protein depletion coupled with separation strategies – microscale solution isoelectrofocusing and 1D SDS-PAGE – followed by reversed-phase separation of tryptic peptides prior to LC–MS/MS. Using this profiling strategy, we routinely identify a large number of proteins over nine orders of magnitude, including a substantial number of proteins at the low ng/mL or lower levels from approximately 300 μL of plasma sample.

Key words

Plasma proteome Low-abundance protein Immunoaffinity depletion Biomarker MicroSol IEF SDS-PAGE LC–MS/MS 

Notes

Acknowledgments

This work was supported in part by National Institutes of Health Grants CA120393 and CA131582, and institutional grants to the Wistar Institute including an NCI Cancer Core Grant (CA10815) and the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health.

References

  1. 1.
    Anderson, N. L., and Anderson, N. G. (2002) The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics 1, 845–867.PubMedCrossRefGoogle Scholar
  2. 2.
    Tirumalai, R. S., Chan, K. C., Prieto, D. A., et al. (2003) Characterization of the low molecular weight human serum proteome. Mol Cell Proteomics 2, 1096–1103.PubMedCrossRefGoogle Scholar
  3. 3.
    Rifai, N., Gillette, M. A. and Carr, S. A. (2006) Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol 24, 971–983.PubMedCrossRefGoogle Scholar
  4. 4.
    Pieper, R., Su, Q., Gatlin, C. L., et al. (2003) Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome, Proteomics 3, 422–432.PubMedCrossRefGoogle Scholar
  5. 5.
    Tang, H. Y., Ali-Khan, N., Echan, L. A. et al. (2005) A novel four-dimensional strategy combining protein and peptide separation methods enables detection of low-abundance proteins in human plasma and serum proteomes, Proteomics 5, 3329–3342.PubMedCrossRefGoogle Scholar
  6. 6.
    Liu, T., Qian, W. J., Gritsenko, M. A. et al. (2006) High dynamic range characterization of the trauma patient plasma proteome, Mol Cell Proteomics 5, 1899–1913.PubMedCrossRefGoogle Scholar
  7. 7.
    Washburn, M. P., Wolters, D., and Yates, J. R., 3rd. (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology, Nat Biotechnol 19, 242–247.PubMedCrossRefGoogle Scholar
  8. 8.
    Shen, Y., Jacobs, J. M., Camp, D. G. et al. (2004) Ultra-high-efficiency strong cation exchange LC/RPLC/MS/MS for high dynamic range characterization of the human plasma proteome, Anal Chem 76, 1134–1144.PubMedCrossRefGoogle Scholar
  9. 9.
    Beer, L. A., Tang, H. Y., Barnhart, K. T., and Speicher, D. W. (2011) Plasma biomarker discovery using 3-D protein profiling coupled with label-free quantitation, Ed. Simpson RJ, Greening DW, Serum/Plasma Proteomics, Methods Mol Biol 728, Humana Press.Google Scholar
  10. 10.
    Omenn, G. S., States, D. J., Adamski, M. et al. (2005) Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database, Proteomics 5, 3226–3245.PubMedCrossRefGoogle Scholar
  11. 11.
    Echan, L. A., Tang, H. Y., Ali-Khan, N., et al. (2005) Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma, Proteomics 5, 3292–3303.PubMedCrossRefGoogle Scholar
  12. 12.
    Echan, L.A., and Speicher, D.W. (2009) Immunoaffinity depletion of high abundance plasma and serum proteins, in The Protein Protocols Handbook, J.M. Walker, Editor. Humana Press, New York, 139–153.CrossRefGoogle Scholar
  13. 13.
    Righetti, P. G., Wenisch, E., Jungbauer, A., et al. (1990) Preparative purification of human monoclonal antibody isoforms in a multi-compartment electrolyser with immobiline membranes, J Chromatogr 500, 681–696.PubMedCrossRefGoogle Scholar
  14. 14.
    Tang, H. Y., and Speicher, D. W. (2005) Complex proteome pre-fractionation using microscale solution isoelectrofocusing, Expert Rev Proteomics 2, 295–306.PubMedCrossRefGoogle Scholar
  15. 15.
    Zuo, X., and Speicher, D. W. (2000) A method for global analysis of complex proteomes using sample pre-fractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis, Anal Biochem 284, 266–278.PubMedCrossRefGoogle Scholar
  16. 16.
    Zuo, X., Echan, L., Hembach, P. et al. (2001) Towards global analysis of mammalian proteomes using sample pre-fractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble and large proteins, Electrophoresis 22, 1603–1615.PubMedCrossRefGoogle Scholar
  17. 17.
    Joo, W. A., and Speicher, D. (2009) Pre-fractionation using microscale solution IEF, Methods Mol Biol 519, 291–304.PubMedCrossRefGoogle Scholar
  18. 18.
    Schwartz, J. C., Senko, M. W., and Syka, J. E. (2002) A two-dimensional quadrupole ion trap mass spectrometer, J Am Soc Mass Spectrom 13, 659–669.PubMedCrossRefGoogle Scholar
  19. 19.
    Douglas, D. J., Frank, A. J., and Mao, D. (2005) Linear ion traps in mass spectrometry, Mass Spectrom Rev 24, 1–29.PubMedCrossRefGoogle Scholar
  20. 20.
    Hu, Q., Noll, R. J., Li, H., et al. (2005) The Orbitrap: a new mass spectrometer, J Mass Spectrom 40, 430–443.PubMedCrossRefGoogle Scholar
  21. 21.
    Makarov, A., Denisov, E., Kholomeev, A., et al. (2006) Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer, Anal Chem 78, 2113–2120.PubMedCrossRefGoogle Scholar
  22. 22.
    Shen, Y., Zhang, R., Moore, R. J. et al. (2005) Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000-1500 and capabilities in proteomics and metabolomics, Anal Chem 77, 3090–3100.PubMedCrossRefGoogle Scholar
  23. 23.
    Eng, J. K., McCormack, A. L., and Yates III, J. R. (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, 976–989.CrossRefGoogle Scholar
  24. 24.
    Tabb, D. L., McDonald, W. H., and Yates, J. R., 3rd. (2002) DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics, J Proteome Res 1, 21–26.PubMedCrossRefGoogle Scholar
  25. 25.
    Rai, A. J., Gelfand, C. A., Haywood, B. C. et al. (2005) HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples, Proteomics 5, 3262–3277.PubMedCrossRefGoogle Scholar
  26. 26.
    Olsen, J. V., de Godoy, L. M., Li, G. et al. (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap, Mol Cell Proteomics 4, 2010–2021.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Hsin-Yao Tang
  • Lynn A. Beer
  • David W. Speicher
    • 1
  1. 1.Molecular and Cellular Oncogenesis ProgramThe Wistar InstitutePhiladelphiaUSA

Personalised recommendations