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

Defining the Campylobacter jejuni interactome by cross-linking mass spectrometry (XL-MS)  (#488)

Ashleigh L Dale 1 2 , Dylan J Harney 1 2 , Isobel J Tenison-Colins 1 2 , Joel A Cain 1 2 , Mark Larance 1 2 , Stuart J Cordwell 1 2 3 4
  1. School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia
  2. Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia
  3. Discipline of Pathology, School of Medical Sciences, University of Sydney, Sydney, NSW, Australia
  4. Sydney Mass Spectrometry, University of Sydney, Sydney, NSW, Australia

Campylobacter jejuni is the leading cause of acute bacterial gastroenteritis in the developed world and human infection is associated with consumption of contaminated poultry, in which the organism is considered an asymptomatic commensal species. Despite the prevalence of infection, the pathogenesis of C. jejuni remains poorly understood. The genome encodes ~1650 proteins, however greater than 50% remain functionally unknown and even less is known about their interactions, or the protein ‘interactome’. Analysis of protein interactions on a global scale is invaluable in developing an understanding of the interconnectivity of biochemical pathways, and ‘interactomics’ facilitated by large-scale, mass spectrometry (MS)-based proteomics has become the method-of-choice for identifying protein-protein interactions (PPIs). Cross-linking mass spectrometry (XL-MS) employs MS-cleavable chemical cross-linkers, such as disuccinimidyl sulfoxide (DSSO), which act to covalently link and stabilise interacting proteins, and allow their unambiguous identification by MS/MS. As a result, XL-MS can be used to predict the function of unknown proteins, validate protein subcellular localisations, refine protein structures, and define significant interaction networks. A novel and optimised approach for XL-MS of total cell lysate and membrane protein enriched fractions using DSSO, size exclusion chromatography and a hybrid MS2-MS3 fragmentation strategy was developed and employed to globally define PPIs in C. jejuni. This enabled the first non-binary and comprehensive analysis of the interactome of this organism. A total of 647 proteins were identified to partake in 868 unique and significant PPIs governed by 2,588 unique Lys-Lys residue contacts. The XL-MS approach successfully covered 39.9% of the predicted proteome of C. jejuni and 49.7% of the proteome as previously identified by ‘bottom-up’ proteomics. Interrogation of the XL-MS dataset yielded known interactions and a large subset of novel interactions, and validated XL-MS as an effective approach to identify, analyse and characterise in vivo PPIs and protein complexes in C. jejuni.