An Integrated Top-Down and Bottom-Up Strategy for Characterization of Protein Isoforms and Modifications

  • Si Wu
  • Nikola Tolić
  • Zhixin Tian
  • Errol W. Robinson
  • Ljiljana Paša-Tolić
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 694)

Abstract

Bottom-up and top-down strategies are two commonly used methods for mass spectrometry (MS) based protein identification; each method has its own advantages and disadvantages. In this chapter, we describe an integrated top-down and bottom-up approach facilitated by concurrent liquid chromatography-mass spectrometry (LC-MS) analysis and fraction collection for comprehensive high-throughput intact protein profiling. The approach employs a high resolution reversed phase (RP) LC separation coupled with LC eluent fraction collection and concurrent on-line MS with a high field (12 T) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer. Protein elusion profiles and tentative modified protein identification are made using detected intact protein mass in conjunction with bottom-up protein identifications from the enzymatic digestion and analysis of corresponding LC fractions. Specific proteins of biological interest are incorporated into a target ion list for subsequent off-line gas-phase fragmentation that uses an aliquot of the original collected LC fraction, an aliquot of which was also used for bottom-up analysis.

Key words

Protein Peptide Proteomics Mass spectrometry LC-MS Top-down Bottom-up FT-ICR MS FTMS Post-translational modification PTM 

Notes

Acknowledgments

Portions of this work were supported by the National Center for Research Resources (RR 018522), the National Institute of Allergy and Infectious Diseases (NIH/DHHS through interagency agreement Y1-AI-4894-01), the National Institute of General Medical Sciences (NIGMS, R01 GM063883), and the U. S. Department of Energy (DOE) Office of Biological and Environmental Research. Work was performed in the Environmental Molecular Science Laboratory, a DOE national scientific user facility located on the campus of Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is a multi-program national laboratory operated by Battelle for the DOE under Contract DE-AC05-76RLO 1830.

References

  1. 1.
    Aebersold, R., Mann, M. (2003) Mass spectromety-based proteomics. Nature 422, 198–207.PubMedCrossRefGoogle Scholar
  2. 2.
    Kelleher, N. L. (2004) Top-down proteomics. Anal. Chem. 76, 197A–203A.PubMedCrossRefGoogle Scholar
  3. 3.
    McLafferty, F. W., Breuker, K., Jin, M., Han, X., Infusini, G., Jiang, H., Kong, X., Begley, T. P. (2007) Top-down MS, a powerful complement to the high capabilities of proteolysis proteomics. FEBS J. 274, 6256–6268.PubMedCrossRefGoogle Scholar
  4. 4.
    Parks, B. A., Jiang, L., Thomas, P. M., Wenger, C. D., Roth, M. J., Ii, M. T., Burke, P. V., Kwast, K. E., Kelleher, N. L. (2007) Top-down proteomics on a chromatographic time scale using linear ion trap Fourier transform hybrid mass spectrometers. Anal. Chem. 79, 7984–7991.PubMedCrossRefGoogle Scholar
  5. 5.
    Siuti, N., Kelleher, N. L. (2007) Decoding protein modifications using top-down mass spectrometry. Nat. Methods 4, 817–821.PubMedCrossRefGoogle Scholar
  6. 6.
    Zabrouskov, V., Whitelegge, J. P. (2007) Increased coverage in the transmembrane domain with activated-ion electron capture dissociation for top-down Fourier-transform mass spectrometry of integral membrane proteins. J. Proteome Res. 6, 2205–2210.PubMedCrossRefGoogle Scholar
  7. 7.
    Zubarev, R. A., Kelleher, N. L., McLafferty, F. W. (1998) Electron capture dissociation of multiply charged proteins cations. A nonergodic process. J. Am. Chem. Soc. 120, 3265–3266.CrossRefGoogle Scholar
  8. 8.
    Coon, J. J., Ueberheide, B., Syka, J. E. P., Dryhurst, D. D., Ausio, J., Shabanowitz, J., Hunt, D. F. (2005) Interpreting the protein language using proteomics. Proc. Natl. Acad. Sci. USA 102, 9463–9468.PubMedCrossRefGoogle Scholar
  9. 9.
    Sharma, S., Simpson, D. C., Tolic, N., Jaitly, N., Mayampurath, A. M., Smith, R. D., Pasa-Tolic, L. (2007) Probing proteomes using capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. J. Proteome Res. 6, 602–610.PubMedCrossRefGoogle Scholar
  10. 10.
    Wu, S., Lourette, N. M., Tolic, N., Zhao, R., Robinson, E. W., Tolmachev, A. V., Smith, R. D., Pasa-Tolic, L. (2009) An integrated top-down and bottom-up strategy for broadly characterizing protein isoforms and modifications. J. Proteome Res. 8, 1347–1357.PubMedCrossRefGoogle Scholar
  11. 11.
    Shen, Y., Tolic, N., Masselon, C., Pasa-Tolic, L., Camp, D. G., Hixson, K. K., Zhao, R., Anderson, G. A., Smith, R. D. (2004) Ultrasensitive proteomics using high-efficiency on-line micro-SPE-nanoLC-nanoESI MS and MS/MS. Anal. Chem. 76, 144–154.PubMedCrossRefGoogle Scholar
  12. 12.
    Tolmachev, A. V., Robinson, E. W., Wu, S., Kang, H., Pasa-Tolic, L., Smith, R. D. (2008) Trapped-ion cell with improved DC potential harmonicity for FT-ICR MS. J. Am. Soc. Mass Spectrom. 19, 586–597.PubMedCrossRefGoogle Scholar
  13. 13.
    Yates, J. R., Eng, J. K., McCormack, A. L., Schieltz, D. (1995) Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal. Chem. 67, 1426–1436.PubMedCrossRefGoogle Scholar
  14. 14.
    Washburn, M. P., Wolters, D., Yates, J. R. (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247.PubMedCrossRefGoogle Scholar
  15. 15.
    Monroe, M. E., Tolic, N., Jaitly, N., Shaw, J. L., Adkins, J. N., Smith, R. D. (2007) VIPER: an advanced software package to support high-throughput LC-MS peptide identification. Bioinformatics 23, 2021–2023.PubMedCrossRefGoogle Scholar
  16. 16.
    LeDuc, R. D., Taylor, G. K., Kim, Y. B., Januszyk, T. E., Bynum, L. H., Sola, J. V., Garavelli, J. S., Kelleher, N. L. (2004) ProSight PTM: an integrated environment for protein identification and characterization by top-down mass spectrometry. Nucleic Acids Res. 32, W340–W345.PubMedCrossRefGoogle Scholar
  17. 17.
    Zamdborg, L., LeDuc, R. D., Glowacz, K. J., Kim, Y. B., Viswanathan, V., Spaulding, I. T., Early, B. P., Bluhm, E. J., Babai, S., Kelleher, N. L. (2007) ProSight PTM 2.0: improved protein identification and characterization for top down mass spectrometry. Nucleic Acids Res. 35, W701–W706.PubMedCrossRefGoogle Scholar
  18. 18.
    Shen, Y., Tolic, N., Hixson, K. K., Purvine, S. O., Anderson, G. A., Smith, R. D. (2008) De novo sequencing of unique sequence tags for discovery of post-translational modifications of proteins. Anal. Chem. 80, 7742–7754.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Si Wu
    • 1
  • Nikola Tolić
    • 1
  • Zhixin Tian
    • 1
  • Errol W. Robinson
    • 1
  • Ljiljana Paša-Tolić
    • 1
  1. 1.Pacific Northwest National LaboratoryRichlandUSA

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