title: A proteomics workflow for quantitative and time-resolved analysis of adaptation reactions of internalized bacteria.
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.
authors: Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Friedrich-Ludwig-Jahn-Strasse 15a, D-17487 Greifswald, Germany.
authors: Henrike
authors: H
authors: Pförtner
authors: Juliane
authors: J
authors: Wagner
authors: Kristin
authors: K
authors: Surmann
authors: Petra
authors: P
authors: Hildebrandt
authors: Sandra
authors: S
authors: Ernst
authors: Jörg
authors: J
authors: Bernhardt
authors: Claudia
authors: C
authors: Schurmann
authors: Melanie
authors: M
authors: Gutjahr
authors: Maren
authors: M
authors: Depke
authors: Ulrike
authors: U
authors: Jehmlich
authors: Vishnu
authors: V
authors: Dhople
authors: Elke
authors: E
authors: Hammer
authors: Leif
authors: L
authors: Steil
authors: Uwe
authors: U
authors: Völker
authors: Frank
authors: F
authors: Schmidt
mesh_terms: D000222:Adaptation, Physiological; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001980:Bronchi / Q000737:chemistry / Q000166:cytology / Q000382:microbiology*; D002247:Carbon Isotopes; D002460:Cell Line; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology / Q000382:microbiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D007553:Isotope Labeling; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D013211:Staphylococcus aureus / Q000737:chemistry* / Q000254:growth & development / Q000378:metabolism; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D002247:Carbon Isotopes; D010455:Peptides; D001120:Arginine; D008239:Lysine