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Metabolomics Analysis in Cecal Samples

ABSTRACT: Targeted LC/MS Metabolomics. Cecal contents were lyophilized overnight and approximately 2.5-5 mg was homogenized in 250 µL of 50% acidified acetonitrile (0.3% formic acid) for targeted LC/MS/MS metabolomics (acylcarnitines, amino acids, organic acids, nucleotides, and CoAs) according to validated, optimized protocols in our previously published study (Previs et al.). Separate aliquots of homogenates were extracted with solvents for each class of metabolites, and then each class was analyzed with a unique LC/MS/MS method to optimize their chromatographic resolution and sensitivity. Quantitation of metabolites in each assay module was achieved using multiple reaction monitoring of calibration solutions and study samples with isotopically-labelled internal standards on an Agilent 1290 Infinity UHPLC/6495 triple quadrupole mass spectrometer. Raw data was processed using Mass Hunter quantitative analysis software (Agilent). Calibration curves (R2 = 0.99 or greater) are either fitted with a linear or a quadratic curve with a 1/X or 1/X2 weighting. Metabolic and Proteomic Defects in Human Hypertrophic Cardiomyopathy Michael J. Previs, Thomas S. O’Leary, Neil B. Wood, Michael P. Morley, Brad Palmer, Martin LeWinter, Jaime Yob, Francis D. Pagani, Christopher Petucci, Min-Soo Kim, Kenneth B. Margulies, Zoltan Arany, Daniel P. Kelly, Sharlene M. Day bioRxiv 2021.08.18.455967; doi: https://doi.org/10.1101/2021.08.18.455967

INSTRUMENT(S): Liquid Chromatography MS - alternating - hilic

PROVIDER: MTBLS14783 | MetaboLights | 2026-06-19

REPOSITORIES: MetaboLights

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			Action	DRS
	a_MTBLS14783_LC-MS_alternating_hilic_metabolite_profiling.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS14783_LC-MS_alternating_normal-phase_v2_maf.tsv	Tabular
	s_MTBLS14783.txt	Txt

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Project description:Depression is a heterogeneous disorder characterized by a wide range of symptoms, including but not restricted to increased anxiety-like behavior, altered stress responsivity, increased depressive like behavior, decreased pleasure seeking, and altered susceptibility to drugs of abuse. Adding another level of complexity to the disease is the fact that individuals differ in their susceptibility to depression. Research done over the past decade has highlighted the contribution of early life adverse experience to this individual differences in vulnerability to depression. Such studies have been done at the clinical as well as the preclinical level, where rodent and primate models of adverse postnatal environment such as Maternal Separation (MS) are used. MS involves separation of the pup from the dam for 3h every day for the first two weeks of postnatal life. The MS model has been characterized to produce long lasting anxiety-like behavior, depressive behavior and altered stress responsivity in adulthood. While several molecular mechanisms have been hypothesized to mediate the long lasting effects of MS, the serotonin 2a receptor is an attractive candidate, given its role in regulating anxiety-like behavior. So we set out to ask if Maternal Separation alters the 5HT2a responses. In order to assay if MS alters the transcriptional targets of the 5Ht2a receptor, we use a drug that stimulates the 5Ht2a receptor, DOI. The experiment involves injecting both control and MS animals with DOI and looking at the transcriptome induced by DOI under control and MS conditions. This would help understand how the adverse early life experience MS, alters the transcriptional response of an adult rat to stimulation at the 5HT2a receptor, which is physiologically seen in conditions of stress. Maternal Separation (MS) was carried out according to standard protocol. Briefly, upon birth the litters were assigned to either the control or the maternal separation group. Pups from the maternal separation litters were separated from their mother every day for a period of 3 hours from postnatal day 2 (p2) to postnatal day 14 (p14) while the control litters were left undisturbed. After p14, the maternally separated pups were left undisturbed and all litters were weaned at postnatal day 30. Experiments on adult control and MS rats were performed at postnatal day 60. We wanted to ask if a history of adverse experience in early life like MS would alter the transcriptional response of the adult rat to the 5Ht2a agonist DOI. In order to measure the transcriptional changes induced by DOI in control and maternally separated animals, 15 rats (7 - Control and 8 - Maternally Separated) were injected i.p. with either saline or 8 mg/kg DOI. The groups included Control (Control rats injected with saline; n=3), DOI (control rats injected with 8 mg/kg DOI; n=4), MS (MS rats injected with saline, n=4) and MS+ DOI (MS rats injected with 8 mg/kg DOI, n=4). Rats were sacrificed 2 hours after the injection by decapitation. The prefrontal cortex was quickly dissected out and stored at -70 till further use. Total RNA from each rat was extracted, labeled with Cy3 and hybridized onto Agilent Custom Rat Array 8X15K (AMADID: G2509F_16352). Each biological replicate was hybridized onto one array making the total number of arrays 15.

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Metabolomics Analysis in Cecal Samples

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