Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Aligning label-based discovery and global DIA validation proteomics to explore bacterial virulence phenotypes (#185)

Lok Man 1 2 , Joel A Cain 1 2 , Stuart J Cordwell 1 2
  1. School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, Australia
  2. Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia

The use of proteomics to inform subsequent biological validation studies requires substantial rigor in the analytical approach to ensure that the most important leads are followed. Our laboratory explores virulence determinants including an N-linked glycosylation (pgl) system and nutrient transporters in the gastrointestinal pathogen, Campylobacter jejuni. C. jejuni is a Gram negative, spiral and micro-aerophilic bacterium with a sequenced genome containing ~1620 genes. Target identification is based on the response of the proteome to environmental conditions that mimic the host, including bile salts, low iron, mucin availability and growth temperatures. The proteomics workflow includes parallel label-based liquid chromatography / tandem mass spectrometry (LC-MS/MS; minimum 3 biological growth replicates) using TMT and / or iTRAQ labelling, and system-wide validation using data independent analysis (DIA-SWATH-MS; minimum duplicate additional biological replicates). We routinely quantify ~80-90% of the predicted C. jejuni NCTC11168 proteome using label-based LC-MS/MS (2 peptides; <1% FDR), and ~65-75% of these proteins can be validated by DIA-SWATH-MS. Here, we will discuss the correlation between large-scale datasets in this biological system and how they facilitate subsequent studies, as well as highlight poorly or non-correlating data. In each case, we show how validated changes in the C. jejuni proteome reflect ‘functional reality’ that can be determined by integrated ‘omics approaches including transcriptomics, metabolomics, lipidomics and lipid A analysis combined with molecular genetics and cellular and in vivo virulence assays. This approach has been employed to characterise functions associated with the pgl system and to identify a nutrient transporter essential for maintaining sulphur import and wild-type human cell virulence phenotypes.