As previously discussed on Thermo Fisher Scientific's Accelerating Proteomic's blog, research into the debilitating inflammatory bowel condition called Crohn’s disease has identified the gut bacterial community as a key factor in the disease’s pathogenesis and continuation. Despite this, there is still no reliable clinical biomarker to speed investigation, monitoring and diagnosis for patients. Because the symptoms of Crohn’s disease are not unique and are shared among many inflammatory bowel diseases, diagnosis frequently requires many different and invasive clinical tests.
In this blog, let's explore how Juste et al. (2014) used the gut microbiota as a starting point in their study exploring the intestinal microbiome for prospective biomarkers1 and how a new platform established by The Crohn's & Colitis Foundation of America (CCFA) may help speed the process towards a potential cure to this disease.
Mining the Gut Microbiota Proteome
Because the intestinal microbial community comprises billions of micro-organisms representing around 1,000 species, cataloging the population is complicated. Many of the mostly anaerobic species are difficult to culture successfully in the lab. Moreover, immunologic techniques require precise antibody specificity, are not practical for multiple analytes, and cannot distinguish easily between protein isoforms. With mass spectrometry-based proteomics techniques, the ability to thoroughly investigate such a wide population has only recently become available. Metagenomic population studies seem to be the best approach to identify key players.
Juste et al. designed their experiment to look at the gut community metaproteome, comparing stool samples from patients with Crohn’s disease (n = 6) with those from healthy volunteers (n = 6). Using two-dimensional differential gel electrophoresis (2D-DIGE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS), they examined differential protein abundance and then quantified the observed changes using selected reaction monitoring (SRM).
First, the researchers extracted bacterial communities from the stool samples to minimize interference from human proteins in subsequent analyses. They used 16S rRNA gene pyrosequencing to confirm that population diversity in the extracted samples matched the diversity found in the stool samples as a whole. Interestingly, the 16S rRNA profile results did not differ according to clinical status.
Once microbial fractions were extracted, the researchers used 2D-DIGE to separate protein signals and examine differential expression between Crohn’s disease and control samples. They discovered 2,007 distinct protein spots across all gels, which they validated and quantified to demonstrate that 93% were unchanged by clinical status. Using LC-MS/MS analysis in an LTQ Orbitrap Discovery hybrid ion trap-Orbitrap mass spectrometer (Thermo Scientific), the researchers analyzed the remaining 7% (n = 141), identifying 89 protein spots as bacterial proteins; of the 141 spots, 53 were increased and 88 were decreased in patients with Crohn’s disease.
For further analysis, Juste and co-authors selected a subset of 59 bacterial proteins, showing that the majority of those increased in abundance were from Bacteroides species, whereas those at lower levels came mostly from the phylum Firmicutes. With functional analysis, the scientists noted that proteins associated with survival and adaption, colonization, metabolic changes and immunostimulation of the host were those that showed differential expression. The researchers confirmed these findings by selecting 13 proteins for micro-LC–SRM quantification using a Dionex Ultimate 3000 system coupled to a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific).
Juste and colleagues note the success of their metaproteomics approach in highlighting bacterial protein signals associated with Crohn’s disease in their small cohort, demonstrating that SRM is a valid tool for quantifying multiple analytes in the complex gut community. The researchers are confident they have identified a valuable method for investigation of biomarkers and therapeutic strategies in this inflammatory bowel disease (IBD).
CCFA Launches IBD Plexus
In May 2015, The CCFA announced a "three-year, $17.5 million grant from The Leona M. and Harry B. Helmsley Charitable Trust to establish an integrated knowledge platform designed to centralize and aggregate patient information – with linked biosamples – across multiple research efforts." This platform, named IBD Plexus, is designed to aid research through better diagnostics, treatments, and cures for Crohn's disease and ulcerative colitis, also known as IBD.
By involving all stakeholders the platform allows for natural collaboration between researchers, patients, and healthcare providers. IBD Plexus has multiple components including a biobank, managed by Fisher BioSerivces, as well as a large data management platform, developed by Deloitte Consulting LLP, to house organize, aggregate and disseminate data. The biobank component of this initiative includes registries to capture clinical, patient-reported, and biosample data (genetic, genomic, microbiomic, etc.). The ultimate plan is that within the timeframe of the grant, clinical data on 40,000 Crohn's and colitis patients will be paired with genomic and microbial profiles from 7,000 patients.
Crohn's disease and ulcerative colitis are painful, it is still unclear why people get IBD, there is no known cure yet, and the incidence of IBD is increasing at an alarming rate in nearly all parts of the world. Through efficient sample and data management facilitated by IBD Plexus, researchers will have the opportunity to take their research to the next level in identifying a cure to this debilitating disease.
To learn more about how a prospective longitudinal study, is making use of mobile devices and cloud-based technology to dramatically cut the time and cost of managing the huge amounts of data, download our eBook about the California Teachers Study (CTS): Next Generation Cohort Studies and Biobanking: How Cloud Technology is Accelerating Translational Research.
Maxwell, Amanda. (2014) Mining the Gut Microbiota Proteome for Crohn’s Disease Biomarkers [blog]. Thermo Scientific.
Crohn's & Colitis Foundation of America. (2015) CCFA Launches Development of World's Largest Research Database for Crohn's Disease and Ulcerative Colitis: First Ever Multi-Dimensional Knowledge Platform to Integrate Scientific, Medical and Patient Data [press release]. PR Newswire.