3rd Annual BMRP Investigator Meeting - Abstract

Quantitative Proteomic Studies of Human Inflammatory Bowel Diseases

Xuhang Li^a, Mark Donowitz, Themos Dassapoulos, Steve Brant, Patricia Ushry and Yuepin Chen

GI Division, Department of Medicine, Johns Hopkins University School of Medicine (Baltimore, Maryland, U.S.A.)

Inflammatory bowel diseases (IBD) are chronic and frequently disabling intestinal inflammatory disorders that affect more than a million Americans and ~0.2% of Western population worldwide.  We have initiated proteomic studies of IBD using biopsies from patients with ulcerative colitis and Crohn’s disease.  Identifying proteins that are differentially expressed or post-translationally modified in IBD vs. normal intestines will increase our current understanding of the pathogenesis of IBD and of the associated diarrhea.  Using 1-D and 2-D gel electrophoresis coupled with differential image gel electrophoresis (DIGE), mass spectrometry (MS), and Western blot screening, our preliminary studies have demonstrated and identified that: 1) a large number of proteins are differentially expressed in IBD intestinal mucosa vs. normal mucosa; and 2) several intestinal epithelial membrane transporters are significantly downregulated in IBD mucosa, suggesting a potential explanation of IBD-associated diarrhea.

Ongoing experiments are expanding to a larger scale and more complicated quantitative proteomics. To increase the chances of identifying changes in relatively low abundant proteins, total cellular proteins were fractionated into several subfractions.  Differential protein expression and modification in each fraction were then analyzed by a combination of a variety of traditional and advanced technologies including 1-D/2-D gel/DIGE/MS technology, or electrospray LC-MS/MS (liquid chromatography MS/MS), from either total mucosa or villus, crypt cells or lamina proprial cells, respectively, isolated by laser capture microdissection (LCM).  Quantitative proteomics approaches being used include 2-D gel/DIGE/MS analysis and O¹⁶/O¹⁸ labeling coupled with LC-MS-MS.  In addition, we are using large-scale targeted screening of IBD mucosa to identify defects in intestinal membrane transporters and their regulatory proteins that are responsible for abnormal Na absorption and Cl secretion, the leading causes for IBD-associated diarrhea.  The ultimate goal is to identify potential diagnostic biomarkers and therapeutic targets for early detection and management/cure of the diseases.

^aPrincipal Investigator