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.