A proteomics workflow for quantitative and time-resolved analysis of adaptation reactions of internalized bacteria Academic Article Article uri icon

Overview

MeSH Major

  • Cell Transformation, Viral
  • Oncogenes
  • Receptors, Cell Surface

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 106cells 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. © 2013 Elsevier Inc.

publication date

  • June 15, 2013

Research

keywords

  • Academic Article

Identity

Digital Object Identifier (DOI)

  • 10.1016/j.ymeth.2013.04.009

PubMed ID

  • 23643866

Additional Document Info

start page

  • 244

end page

  • 250

volume

  • 61

number

  • 3