This article was originally published in Proteomics 2010, 10, 2801–2811, DOI 10.1002/pmic.201000045

Time-resolved quantitative proteome profiling of

host–pathogen interactions: The response of

Staphylococcus aureus RN1HG to internalisation by

human airway epithelial cells

Frank Schmidt, Sandra S. Scharf, Petra Hildebrandt, Marc Burian, Jorg Bernhardt,Vishnu Dhople, Julia Kalinka, Melanie Gutjahr, Elke Hammer and Uwe Volker

Keywords:

Host–pathogen interaction / In vivo proteomics / Microbiology / Pulse-chase SILAC / S. aureus /

S9 human bronchial epithelial cells

Staphylococcus aureus is a versatile Gram-positive pathogen

that gains increasing importance due to the rapid spreading

of resistances. Functional genomics technologies can provide

new insights into the adaptational network of this bacterium

and its response to environmental challenges. While func-

tional genomics technologies, including proteomics, have

been extensively used to study these phenomena in shake

flask cultures, studies of bacteria from in vivo settings lack

behind. Particularly for proteomics studies, the major

bottleneck is the lack of sufficient proteomic coverage for low

numbers of cells. In this study, we introduce a workflow that

combines a pulse-chase stable isotope labelling by amino

acids in cell culture approach with high capacity cell sorting,

on-membrane digestion, and high-sensitivity MS to detect

and quantitatively monitor several hundred S. aureus proteins

from a few million internalised bacteria. This workflow has

been used in a proof-of-principle experiment to reveal chan-

ges in levels of proteins with a function in protection against

oxidative damage and adaptation of cell wall synthesis in

strain RN1HG upon internalisation by S9 human bronchial

epithelial cells.Quantitative analysis of changes in the protein level of inter-

nalised S. aureus RN1HG.

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Proteomics Clin. Appl. 2010, 4, 847–866 861