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.

Bimodal plasma metabolomics strategy identifies novel inflammatory metabolites in inflammatory bowel diseases.

INTRODUCTION: Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) characterized by variable phenotypes. Metabolites are signatures of biochemical activity that can reveal unknown pathogenic pathways. We employed untargeted mass spectrometry (MS) based metabolomics to identify novel inflammatory mechanisms in IBD and a targeted assay to quantify metabolites of the auto-immunomodulating kynurenine pathway (KP) in IBDs and health. MATERIALS AND METHODS: Metabolome analysis of CD, UC, and control plasmas was performed on a Liquid Chromatography (LC)-MS/MS system. KP metabolites quinolinic acid (QA) and picolinic acid (PA) were quantified by gas chromatography/MS. RESULTS: Nineteen UC, 25 CD, and 9 control plasmas were analyzed: 34 metabolites exhibited abundance profiles associated with CD by global metabolome analysis (P≤0.05, false discovery rate q≤0.01). Notably, inflammatory-implicated metabolites angiotensin IV (P=0.049, q<0.001), diphthamide (P=0.018, q<0.001), and GM3 gangliosides (P<0.001, q<0.001) were increased in CD. By targeted kynurenine metabolites assay, QA (73.53 ng/mL ± 23.40 SD) and combined kynurenine metabolites (CKM) were increased in CD (120.19 ± 39.71) compared to controls (QA 50.14 ± 15.04; P<0.01; CKM 92.73 ± 26.30; P<0.01). CD QA positively correlated with CDAI (r=0.85; P<0.01), CRP (r=0.46; P=0.01), and ESR (r=0.42; P=0.03), while CKMs correlated with CDAI (r=0.615; P<0.01) and CRP (r=0.615; P=0.02). CONCLUSIONS: Associations of angiotensin IV, diphthamide, and GM3 gangliosides with CD implicate novel pathways in activating a Th1/Th17 inflammatory profile. Increased QA concentrations in CD may indicate a defective auto-immunomodulation mechanism.