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.
Kelly
John F
JF
Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada.
Ding
Wen
W
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005604:Francisella tularensis / Q000378:metabolism*; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D036103:Nanotechnology; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry / Q000379:methods*
D001426:Bacterial Proteins; D010455:Peptides