Disease-specific proteins, including low-mass peptides, can be difficult to detect amongst a diverse range of concentrations present for plasma proteins. Combined with the immense extent of human and disease variation and the challenges facing the development of sensitive and specific differentiators; developing diagnostics suitable for clinical use is a formidable task. Discovery phase quantitative approaches entail the differentiation of as many peptides as possible (rather than the identification of all proteins) from LC-MS experiments. The development of targeted, quantitative approaches that provide accurate and statistically reliable quantitative outcomes for multi-site studies may provide a critical bridge to establishing validity of individual or panels of biomarkers.
Inflammatory bowel disease (IBD) is a life-long relapsing and remitting inflammatory disorder primarily affecting the gastrointestinal tract and is mainly subdivided into Crohn’s disease (CD) and ulcerative colitis (UC).1 Current treatment regimen aims to dampen the vigorous inflammation without a cure. The importance of an accurate diagnosis is paramount as the prognosis for CD and UC differ markedly, although a definitive diagnosis is difficult to achieve as clinical manifestations are very similar. Protein markers may strengthen the discrimination of IBD subtypes and provide prognostic information. This study has incorporated the techniques of low mass partitioning, LC-MS/MS and label–free quantitation for a discovery based survey of unique peptides and proteins able to distinguish between our phenotypes. Selected candidates have then been further evaluated in fresh cohorts comprising of 109 patients using multiple light and heavy synthetic peptides to determine absolute quantities within those phenotypes. Antibody detection using Westerns has also been carried out. Discussed will be the identification of unique differentiators for application to diagnosis of IBD’s.