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HRMS1 DIA and DDA of Lyso-IP inputs from mouse gut epithelium and stem cells

ABSTRACT: These mouse gut lysates contained three sets, with Set A including 21 DDA experiments and 21 DIA experiments, Set C including 24 DDA and 24 DIA experiments, and Set G including 27 DDA RAWs and 26 DIA RAWs (one of the Set G DDA experiments was run twice). Experiment Set A was intended to compare lysosomal compositions among ad libitum feeding, 24h fasted, and 24h fasted and re-fed conditions. Experiment Set C compared lysosomal compositions between germ-free and conventional microbiome states. Experiment Set G compared ad libitum and 24h fasted conditions within stem cells. In each case, these RAWs represent the inputs to lyso-IP, not the lysosomally-enriched samples that are produced from lyso-IP. Sample preparation was performed using the S-TRAP method, with MMTS (methyl methanethiosulfonate) alkalyting free Cys side chains and trypsin digesting C-terminal to lysines and arginines. DDA experiments collected spectra for all 120 minutes of a two hour run, while DIA experiments using the high resolution MS1 (HRMS1) technique collected scans at two different FAIMS compensation voltages for 75 minutes during each 105 minute run, with scan events ending when the B solvent of the gradient reached 45%.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Omer Yilmaz John P. LaCava

PROVIDER: MSV000095444 | MassIVE | Fri Jul 26 01:41:00 BST 2024

SECONDARY ACCESSION(S): PXD054265

REPOSITORIES: MassIVE

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Project description:Delayed access to feed following hatching has been associated with reduction in growth performance in chicks. However, the gene networks within the hypothalamus that regulate feed intake and metabolism, and the effects of fasting on those pathways are not well understood. The present experiment evaluated global hypothalamic gene expression in neonatal chicks using the Arizona Gallus gallus 20.7K Oligo Array v1.0 to elucidate genes and pathways regulated by feeding, fasting, and refeeding. Ten groups of chicks were sampled over four days post-hatch, including: control (at hatch), 24h fed, 24h fasted, 48h fed, 48h fasted, 48h fasted then 4h refed, 72h fed, 48h fasted then 24h refed, 96h fed, and 48h fasted then 48h refed. Non-esterified fatty acids were elevated, and triglycerides were decreased in fasted chicks at 24 and 48h, indicating that fasting induced physiological changes. Hypothalamic samples were collected from 16 chicks per group, and total RNA was extracted and pooled for hybridization (n=4). Expression patterns of selected genes were confirmed by quantitative real-time PCR. Two-fold differences in gene expression were detected in 1,272 genes between treatment groups, and of those, 119 genes were significantly (P<0.05) different. Assignment of gene ontology terms to the significant genes resulted in 34 different categories of biological processes, with 24% of genes participating in signal transduction, transport, or metabolic processes. Genes that were upregulated during fasting (and confirmed by qPCR) include FK506BP51, which is involved in the formation of steroid receptor complexes, and deiodinase type II, which is responsible for converting the thyroid hormone T4 into T3. Confirmed genes, downregulated due to fasting, included proopiomelanocortin and fatty acid binding protein 7. Other regulated genes were identified that play a role in feeding and obesity in other species, such as relaxin 3 and adrenergic receptor-β2. Further analysis of differentially regulated genes could provide new information regarding the role of the hypothalamus in feed intake and metabolism. Keywords: Metabolic perturbation

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HRMS1 DIA and DDA of Lyso-IP inputs from mouse gut epithelium and stem cells

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