Low Mass Blood Peptides Discriminative of Inflammatory Bowel Disease (IBD) Severity: A Quantitative Proteomic Perspective

Breakdown of the protective gut barrier releases effector molecules and degradation products into the blood stream making serum and plasma ideal as a diagnostic medium. The enriched low mass proteome is unexplored as a source of differentiators for diagnosing and monitoring inflammatory bowel disease (IBD) activity, that is less invasive than colonoscopy. Differences in the enriched low mass plasma proteome (<25 kDa) were assessed by label-free quantitative mass-spectrometry. A panel of marker candidates were progressed to validation phase and “Tier-2” FDA-level validated quantitative assay. Proteins important in maintaining gut barrier function and homeostasis at the epithelial interface have been quantitated by multiple reaction monitoring in plasma and serum including both inflammatory; rheumatoid arthritis controls, and non-inflammatory healthy controls; ulcerative colitis (UC), and Crohn''s disease (CD) patients. Detection by immunoblot confirmed presence at the protein level in plasma. Correlation analysis and receiver operator characteristics were used to report the sensitivity and specificity. Peptides differentiating controls from IBD originate from secreted phosphoprotein 24 (SPP24, p = 0.000086, 0.009); whereas those in remission and healthy can be differentiated in UC by SPP24 (p = 0.00023, 0.001), α-1-microglobulin (AMBP, p = 0.006) and CD by SPP24 (p = 0.019, 0.05). UC and CD can be differentiated by Guanylin (GUC2A, p = 0.001), and Secretogranin-1 (CHGB p = 0.035). Active and quiescent disease can also be differentiated in UC and CD by CHGB (p ≤ 0.023) SPP24 (p ≤ 0.023) and AMBP (UC p = 0.046). Five peptides discriminating IBD activity and severity had very little-to-no correlation to erythrocyte sedimentation rate, C-reactive protein, white cell or platelet counts. Three of these peptides were found to be binding partners to SPP24 protein alongside other known matrix proteins. These proteins have the potential to improve diagnosis and evaluate IBD activity, reducing the need for more invasive techniques. Data are available via ProteomeXchange with identifier PXD002821.Inflammatory bowel disease (IBD)¹ is a life-long relapsing and remitting inflammatory disorder primarily affecting the gastrointestinal tract and can be subdivided into the main groups of Crohn''s disease (CD) and ulcerative colitis (UC) (1). Current treatment focuses on reducing and controlling inflammation. There is no cure and the majority of IBD patients remain under medical care and management for life. With increasing prevalence around the world, clinical assays that can provide accurate diagnosis, discrimination between CD and UC, and determination of disease activity are being sought to achieve effective treatment and management. The clinical presentations of both subtypes are similar and invasive diagnostic investigations, specifically colonoscopy and histopathological evaluation of the inflamed gut wall, remains the gold standard for diagnosis and assessment of activity (2–5). Current diagnostic antibody markers such as anti-saccharomyces cerevisiae antibody (ASCA) and peri-nuclear anti-neutrophil cytoplasmic antibody (P-ANCA) or combinations of genetic susceptibility markers and serological markers provide increased specificity (6–10). Despite this, acute phase proteins such as C-reactive protein (CRP), fecal calprotectin in addition to the erythrocyte sedimentation rate (ESR) and other clinical activity indicators are more typically used in practice to monitor disease progression in addition to colonoscopy (11). Unbiased discovery in patient plasma samples has the potential to capture both the reactive pathways that result in symptoms as well as identify novel causal proteins that may have initiated disease onset and the biological switch to autoimmune complications of IBD (12, 13). The regulation of homeostasis between the intestinal epithelial cells, mucosal surface, and the immune system that contribute to exacerbated inflamed response are less well characterized and would benefit from the posteriori knowledge of the global “omics” approach to explore emerging causal and reactive proteins and peptides for further validation. Discovery of new protein markers through proteomic technology has already expanded the knowledge of IBD (14–19) and can be used to improve the diagnostic accuracy, long-term management, and treatment of a host of different diseases (20, 21).We have specifically focused on the differential protein profiles of 1–25 kDa fraction between IBD and healthy human plasma samples. Such partitioning of proteins enabled powerful enrichment of low mass and poorly abundant proteins (22). Using a shotgun proteomic approach, this large scale survey of proteins has highlighted the increase in inflammatory and acute phase proteins that are known to plague the illness and in addition has revealed novel peptides and proteins that can be used to discriminate IBD from controls, and UC from CD. These proteins have been investigated further using accurate and sensitive quantitative techniques of multiple reaction monitoring (MRM) for low-concentration peptides (23) applicable to verification phase Tier 2 multiplexed MRM assay development within the FDA-National Cancer Institute (NCI) biomarker pipeline (24). The on-column amounts of each protein from this biomarker panel were evaluated for individual samples, and Western blots have also been used to confirm presence.