Speaker
Description
Myo-inositol (MI) is a biologically important cyclitol involved in diverse physiological processes in animals, yet its role as a substrate for intestinal microbial communities remains poorly understood. Although genomic studies have suggested that gut microorganisms possess pathways associated with MI degradation, experimental evidence demonstrating active MI utilization within the poultry intestinal microbiome is still limited. Understanding microbial substrate assimilation requires approaches capable of linking metabolic activity to specific microbial proteins and taxa. In this study, protein stable isotope probing (Protein-SIP) combined with high-resolution metaproteomics was applied to investigate anaerobic MI metabolism in the intestinal microbiota of laying hens.
Ileal and caecal digesta samples were incubated under anaerobic conditions in a customized minimal medium containing uniformly 13C-labelled MI as the primary carbon source. Samples collected at multiple time points were subjected to protein extraction and LC-MS/MS analysis to monitor isotope incorporation into microbial proteins. Metabolomic analysis of labelled metabolites was performed using nuclear magnetic resonance (NMR) spectroscopy to complement protein-based measurements. Identification of 13C-labelled peptides and assignment of microbial protein origins were performed using the SIPROS 4 workflow.
The computational analysis of Protein-SIP datasets represents a considerable challenge due to the increased search complexity associated with isotope-labelled peptides and the large volume of mass spectrometry data generated. In this study, 387 LC-MS/MS raw files with a combined size of approximately 566 GB were processed using SIPROS 4 through JupyterLab on the BinAC1 high-performance computing system within the bwHPC infrastructure. The available computational resources enabled efficient processing of the complete dataset and facilitated reproducible analysis of large-scale metaproteomic measurements that would have been difficult to perform using conventional local computing environments.
Protein-SIP analysis demonstrated active incorporation of 13C from MI into microbial proteins involved in energy production and conversion, carbohydrate transport and metabolism, and translation-related processes. Distinct metabolic patterns were observed between intestinal compartments, with the caecal microbiota showing earlier and stronger MI utilization compared with the ileal community. Taxonomic assignment of labelled proteins identified Megamonas as a major contributor to MI metabolism in the caecum, whereas isotope incorporation in the ileum was distributed across multiple bacterial groups, indicating differences in ecological niches and metabolic specialization along the intestinal tract. For the first time, this research highlights Gallibacterium as a candidate MI metabolizer in ileum.
This study provides the first experimental characterization of anaerobic MI metabolism within the poultry intestinal microbiome using isotope-resolved metaproteomics. Beyond revealing previously uncharacterized microbial functions, this work demonstrates the importance of scalable computational resources for analysing complex multi-gigabyte proteomic datasets. The integration of advanced mass spectrometry, stable isotope probing, and HPC-enabled data processing provides a framework for investigating microbial metabolism at community scale and highlights the role of shared research infrastructures such as bwHPC in enabling data-intensive life science research.