| Literature DB >> 23643866 |
Henrike Pförtner1, Juliane Wagner, Kristin Surmann, Petra Hildebrandt, Sandra Ernst, Jörg Bernhardt, Claudia Schurmann, Melanie Gutjahr, Maren Depke, Ulrike Jehmlich, Vishnu Dhople, Elke Hammer, Leif Steil, Uwe Völker, Frank Schmidt.
Abstract
The development of a mass spectrometric workflow for the sensitive identification and quantitation of the kinetics of changes in metaproteomes, or in particular bacterial pathogens after internalization by host cells, is described. This procedure employs three essential stages: (i) SILAC pulse-chase labeling and infection assay; (ii) isolation of bacteria by GFP-assisted cell sorting; (iii) mass spectrometry-based proteome analysis. This approach displays greater sensitivity than techniques relying on conventional cell sorting and protein separation, due to an efficient combination of a filtration-based purification and an on-membrane digestion. We exemplary describe the use of the workflow for the identification and quantitation of the proteome of 10⁶ cells of Staphylococcus aureus after internalization by S9 human bronchial epithelial cells. With minor modifications, the workflow described can be applied for the characterization of other host-pathogen pairs, permitting identification and quantitation of hundreds of bacterial proteins over a time range of several hours post infection.Entities:
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Year: 2013 PMID： 23643866 DOI： 10.1016/j.ymeth.2013.04.009
Source DB: PubMed Journal: Methods ISSN： 1046-2023 Impact factor: 3.608