Using 2D-LC-MS/MS to Identify Francisella tularensis Peptides in Extracts from an Infected Mouse Macrophage Cell Line

  • John F. Kelly
  • Wen Ding
Part of the Methods in Molecular Biology™ book series (MIMB, volume 439)


Two dimensional nano-high-performance liquid chromatography (nanoHPLC) coupled directly to a high-resolution tandem mass spectrometer (2D-nLC-MS/MS) is an excellent method for analyzing very complex peptide mixtures, especially when the quantity of sample available for analysis is severely limited. We describe here a relatively simple 2D-nLC-MS/MS approach that we often use to characterize complex peptide mixtures, such as those produced by the proteolytic digestion of protein extracts. A peptide mixture is resolved in the first dimension by stepped elution from a strong cation exchange (IEC) column and in the second dimension by reverse phase (RP) nanoHPLC chromatography prior to electrospray ionization. The peptide ions are analyzed by automatic tandem mass spectrometry in a hybrid quadrupole time-of-flight mass spectrometer (Q-TOF). In this chapter, we illustrate this approach by way of an example featuring analyses of peptides extracted from a mouse macrophage cell line infected with the hve vaccine strain of Francisella tularensis.


two-dimensional liquid chromatography-tandem mass spectrometry Francisella tularensis automated database searching MHC mouse macrophage cells 



We thank Dr. Wayne Conlan (NRC-IBS) and Dr. Anders Sjöstedt (Umeå U.) for their guidance and support during this work. We also thank Dr. John Nash, Simon Foote, and Ken Chan (all from NRC-IBS) for their assistance with the informatics associated with this project.


  1. 1.
    Brockman A (1999) Rapid Communications in Mass Spectrometry 13:1024–1030Google Scholar
  2. 2.
    Flyer D. (2002) Infection and Immunity 70:2926–2932.Google Scholar
  3. 3.
    Hughes C (2001) Presented at the 49th American Society for Mass Spectrometry conference, Chicago, May 27–31Google Scholar
  4. 4.
    4. Link AJ, Eng J, Schieltz DM, Carmack E, Mize GJ, Morris DR, Garvik BM, Yates JR (1999) Direct analysis of protein complexes using mass spectrometry. Nature Biotechnology 17:676–682CrossRefPubMedGoogle Scholar
  5. 5.
    5. Washburn MP, Wolters D, Yates JR (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature Biotechnology 19:242–247CrossRefPubMedGoogle Scholar
  6. 6.
    6. Washburn MP, Ulaszek R, Deciu C, Schieltz DH, Yates JR (2002) Analysis of quantitative proteomics data generated via multidimensional protein identification technology. Analytical Chemistry 74:1650–1657CrossRefPubMedGoogle Scholar
  7. 7.
    7. Washburn MP, Ulaszek RR, Yates JR (2003) Reproducibility of quantitative proteomic analysis of complex biological mixtures by multidimensional protein identification technology. Analytical Chemistry 75:5054–5061CrossRefPubMedGoogle Scholar
  8. 8.
    8. Lubman DM, Kachman MT, Wang H, Gong S, Yan F, Hamler RL, O'Neil KA, Buchanan NS, Barder TJ (2002) Two-dimensional liquid separations-mass mapping of proteins from human cancer cell lysates. Journal of Chromatography B 782:183–196CrossRefGoogle Scholar
  9. 9.
    9. Kachman MT, Wang H, Schwartz DR, Cho KR, Lubman DM (2002) A 2-D liquid separations/ mass mapping method for interlysate comparison of ovarian cancers. Analytical Chemistry 74:1779–1791CrossRefPubMedGoogle Scholar
  10. 10.
    10. Hamler RL, Zhu K, Buchanan NS, Kreunin P, Kachman MT, Miller FR, Lubman DM (2004) A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomics studies of breast cancer and biomarker identification. Proteomics 4:562–577CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • John F. Kelly
    • 1
  • Wen Ding
    • 1
  1. 1.Institute for Biological SciencesNational Research CouncilOttawaCanada

Personalised recommendations