Profiling the low-mass serum proteome of the behavioural phenotypes of Crohn’s disease

Abstract

Background: Stricturing and fistulizing intestinal complications are largely responsible for the significant morbidity of Crohn’s disease (CD). The inability to predict the development of these complicated phenotypes of CD (CCD) remains the bottleneck to evaluating the efficacy of early escalation therapies, which could change the natural history of CD. Proteomic methodologies may be able to identify important mechanisms in these pathologies. This thesis comprised a series of studies conducted under the framework of the biomarker development pipeline to examine the low-mass serum proteome of the behavioural phenotypes of CD.

Methods: Untargeted and targeted Mass Spectrometry (MS) was used for global metabolite profiling and quantification of immunoregulating Kynurenine Pathway (KP) metabolites in proof-of-principle metabolomics studies. In a discovery phase proteomics study, in-solution electrophoresis was used to enrich the low-mass (<25kDa) serum fraction of CD behavioural phenotypes for untargeted MS analysis. Elements affecting accuracy in Multiple Reaction Monitoring (MRM) MS assays were evaluated in research assay optimisation phase study, and MRM and immunoblotting were used in qualification phase proteomics studies to quantify discovery phase-identified biomarker candidates in cross-sectional and longitudinal cohorts.

Results: Proof-of-principle metabolomic studies demonstrated the ability of MS to identify unique plasma profiles between distinct pathologies of intestinal inflammation. Th1/17 inflammatory metabolites Angiotensin IV, diphthamide and GM3 gangliosides were associated with CD and KP metabolite Quinolinic acid was increased in CD and correlated with biochemical and clinical measures of disease activity. In discovery phase proteomics study, a low-mass serum profile typified by an overabundance of epithelial component proteins was identified in CCD. In research assay optimisation, peptide-centric matrix effects that caused deleterious effects on quantitative accuracy in MRM assays were identified, and a novel Reverse-Polynomial Dilution (RPD) calibration technique that reduced error in multiplexed MRM assays was established. Finally, a serological biomarker candidate panel with discriminative ability for intestinal complications in CD was demonstrated in qualification phase.

Conclusions: An enrichment of serological epithelial component proteins in CCD was identified in this work that can classify CCD against intestinal and Th1/17 systemic inflammation controls. These proteins may be serological biomarkers of transmural intestinal tissue integrity that could predict progression to CCD.