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