Urinary Proteome Analysis of Irritable Bowel Syndrome (IBS) Symptom Subgroups

Irritable bowel syndrome (IBS) is a functional gastrointestinal
(GI) disorder characterized by chronic abdominal pain associated with
alterations in bowel function. Given the heterogeneity of the symptoms,
multiple pathophysiologic factors are suspected to play a role. We
classified women with IBS into four subgroups based on distinct symptom
profiles. In-depth shotgun proteomic analysis was carried out to profile
the urinary proteomes to identify possible proteins associated with
these subgroups. First void urine samples with urine creatinine level ≥100
mg/dL were used after excluding samples that tested positive for blood.
Urine from 10 subjects representing each symptom subgroup was pooled
for proteomic analysis. The urine proteome was analyzed by liquid
chromatography–tandem mass spectrometry (LC–MS/MS) using
a data-independent method known as Precursor Acquisition Independent
From Ion Count (PAcIFIC) that allowed extended detectable dynamic
range. Differences in protein quantities were determined by peptide
spectral counting followed by validation of select proteins with ELISA
or a targeted single reaction monitoring (LC–SRM/MS) approach.
Four IBS symptom subgroups were selected: (1) Constipation, (2) Diarrhea
+ Low Pain, (3) Diarrhea + High Pain, and (4) High Pain + High Pychological
Distress. A fifth group consisted of Healthy Control subjects. From
comparisons of quantitative spectral counting data among the symptom
subgroups and controls, a total of 18 proteins that showed quantitative
differences in relative abundance and possible physiological relevance
to IBS were selected for further investigation. Three of the 18 proteins
were chosen for validation by either ELISA or SRM. An elevated expression
of gelsolin (GSN) was associated with the high pain groups. Trefoil
Factor 3 (TFF3) levels were higher in IBS groups compared to controls.
In this study, the IBS patients subclassified by predominant symptoms
showed differences in urine proteome levels. Proteins showing distinctive
changes are involved in homeostasis of intestinal function and inflammatory
response. These findings warrant future studies with larger, independent
cohorts to enable more extensive assessment and validation of urinary
protein markers as a diagnostic tool in adults with IBS.