Using 2D-LC-MS/MS to identify Francisella tularensis peptides in extracts from an infected mouse macrophage cell line: Methods Mol.Biol.

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TypeArticle
Journal titleMethods Mol.Biol.
Volume439
Pages257267; # of pages: 11
Subjectanalysis; Animals; bacterial; Bacterial Proteins; Canada; cell; Cell Line; chemistry; Chromatography; Chromatography,High Pressure Liquid; complex; COMPLEXES; Digestion; Electrospray; exchange; Francisella tularensis; HIGH-RESOLUTION; Ions; isolation & purification; Macrophages; mass spectrometry; MASS-SPECTROMETRY; metabolism; method; Methods; Mice; Microbiology; Mixture; Nanotechnology; peptide; Peptides; PHASE; protein; Proteins; STRAIN; Tandem Mass Spectrometry
AbstractTwo 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
Publication date
LanguageEnglish
AffiliationNRC Institute for Biological Sciences; National Research Council Canada
Peer reviewedNo
NRC numberKELLY2008
NPARC number9363320
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Record identifierd4daa6dd-fe97-460b-b7b5-4aad45a06e79
Record created2009-07-10
Record modified2016-05-09
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