11-Plex DiLeu Isobaric Labeling Enables Quantitative Assessment of Brain Region Protein Association Networks Impacted by the Gut Microbiome.

Gut microbiota can regulate host brain functions and influence various physiological and pathological processes through the brain-gut axis. To systematically elucidate the intervention of different gut environments on different brain regions, we implemented an integrated approach that combines 11-plex DiLeu isobaric tags with a "BRIDGE" normalization strategy to comparatively analyze the proteome of six brain regions in germ-free (GF)- and conventionally raised (ConvR)-mice. A total of 5945 proteins were identified and 5656 were quantifiable, while 1906 of them were significantly changed between GF- and ConvR-mice; 281 proteins were filtered with FC greater than 1.2 in at least one brain region, of which heatmap analysis showed clear protein profile disparities, both between brain regions and gut microbiome conditions. Gut microbiome impact is most overt in the hypothalamus and the least in the thalamus region. Collectively, this approach allows an in-depth investigation of the induced protein changes by multiple gut microbiome environments in a brain region-specific manner. This comprehensive proteomic work improves the understanding of the brain region protein association networks impacted by the gut microbiome and highlights the critical roles of the brain-gut axis.

Integrated Label-Free and 10-Plex DiLeu Isobaric Tag Quantitative Methods for Profiling Changes in the Mouse Hypothalamic Neuropeptidome and Proteome: Assessment of the Impact of the Gut Microbiome.

Gut microbiota can regulate host physiological and pathological status through gut-brain communications or pathways. However, the impact of the gut microbiome on neuropeptides and proteins involved in regulating brain functions and behaviors is still not clearly understood. To address the problem, integrated label-free and 10-plex DiLeu isobaric tag-based quantitative methods were implemented to compare the profiling of neuropeptides and proteins in the hypothalamus of germ-free (GF)- vs conventionally raised (ConvR)-mice. A total of 2943 endogenous peptides from 63 neuropeptide precursors and 3971 proteins in the mouse hypothalamus were identified. Among these 368 significantly changed peptides (fold changes over 1.5 and a p-value of <0.05), 73.6% of the peptides showed higher levels in GF-mice than in ConvR-mice, and 26.4% of the peptides had higher levels in ConvR-mice than in GF-mice. These peptides were mainly from secretogranin-2, phosphatidylethanolamine-binding protein-1, ProSAAS, and proenkephalin-A. A quantitative proteomic analysis employing DiLeu isobaric tags revealed that 282 proteins were significantly up- or down-regulated (fold changes over 1.2 and a p-value of <0.05) among the 3277 quantified proteins. These neuropeptides and proteins were mainly involved in regulating behaviors, transmitter release, signaling pathways, and synapses. Interestingly, pathways including long-term potentiation, long-term depression, and circadian entrainment were involved. In the present study, a combined label-free and 10-plex DiLeu-based quantitative method enabled a comprehensive profiling of gut microbiome-induced dynamic changes of neuropeptides and proteins in the hypothalamus, suggesting that the gut microbiome might mediate a range of behavioral changes, brain development, and learning and memory through these neuropeptides and proteins.