Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA209196

Local Sample ID:	2020-08-17_Zang-43_Box2Redo-2_POS
Subject ID:	SU002264
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	10-week and 24-month
Gender:	Male

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Subject:

Subject ID:	SU002264
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	10-week and 24-month
Gender:	Male

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
2020-08-17_Zang-43_Box2Redo-2_POS	SA209196	FL025531	Young	Age Group
2020-08-17_Zang-43_Box2Redo-2_POS	SA209196	FL025531	peptide	Treatment

Collection:

Collection ID:	CO002257
Collection Summary:	Wild-type (WT) C57BL/6 mice were used in this study. Endotoxemia was induced in young (10-week) and aged (24-week) male mice by lipopolysaccharide (LPS). Based on published results as well as observations in our laboratory, male and female mice showed significantly different susceptibility to systemic symptoms in sepsis models. Thus, male but not female mice were chosen for the experiments presented in this report. LPS was administered intraperitoneally (i.p.), and mice were weighed individually to determine the exact amount of LPS (MilliporeSigma, Burlington, MA; catalog number L3012) required to achieve the doses indicated in the figure legends. Sterile endotoxin-free PBS was used as a vehicle control in sham groups. In some experiments, Beclin-1-activating peptide (TB peptide), synthesized according to a published sequence24 by NonoPep (Shanghai, China), was administered i.p. at a dose of 16 mg/kg in 100μl of PBS 30 minutes post LPS-challenge. Heart tissue was harvested 18 hours post LPS challenge.
Collection Protocol Filename:	Zang_Protocol.docx
Sample Type:	Heart
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002276
Treatment Summary:	Endotoxemia was induced in young (10-week) and aged (24-week) male mice by lipopolysaccharide (LPS). Based on published results as well as observations in our laboratory, male and female mice showed significantly different susceptibility to systemic symptoms in sepsis models. Thus, male but not female mice were chosen for the experiments presented in this report. LPS was administered intraperitoneally (i.p.), and mice were weighed individually to determine the exact amount of LPS (MilliporeSigma, Burlington, MA; catalog number L3012) required to achieve the required doses. Sterile endotoxin-free PBS was used as a vehicle control in sham groups. Consistent with literature and as expected, we observed that older mice were more susceptible to the toxic effects induced by LPS. 24-month-old (aged) mice showed impaired cardiac function but were able to survive when receiving LPS challenged at 1mg/kg. However, greater fatality was observed when LPS dose was increased to 3 mg/kg. In 10-week-old (young adult) mice, 3 mg/kg LPS triggered heart dysfunction without impact on survival, whereases at 10 mg/kg, we observed significant LPS-induced fatality in the group. Due to the different sensitivities to LPS between the aged and young adult mice, we were not able to choose a universal dose of LPS to induce cardiac dysfunction and to perform follow up analysis in both groups. Therefore, we used the physiological function of the heart as a base for comparison in the studies performed in this report. Our previous research provided evidence that stimulating beclin-1 dependent autophagy improves cardiac performance during endotoxemia in young adult mice, and thus Beclin-1-activating peptide (TB peptide) holds a promising therapeutic potential for sepsis. In this report, we examined whether TB-peptide exerts a similar protective effect on aged animals under the same condition. In our experimental setting, sham or LPS challenge was administered to groups of 24-month-old and 10-week-old mice followed by treatment with TB-peptide, administered i.p. at a dose of 16 mg/kg in 100μl of PBS 30 minutes post LPS-challenge. Heart tissue was collected 18 hours post LPS challenge.

Treatment ID:

TR002276

Treatment Summary:

Endotoxemia was induced in young (10-week) and aged (24-week) male mice by lipopolysaccharide (LPS). Based on published results as well as observations in our laboratory, male and female mice showed significantly different susceptibility to systemic symptoms in sepsis models. Thus, male but not female mice were chosen for the experiments presented in this report. LPS was administered intraperitoneally (i.p.), and mice were weighed individually to determine the exact amount of LPS (MilliporeSigma, Burlington, MA; catalog number L3012) required to achieve the required doses. Sterile endotoxin-free PBS was used as a vehicle control in sham groups. Consistent with literature and as expected, we observed that older mice were more susceptible to the toxic effects induced by LPS. 24-month-old (aged) mice showed impaired cardiac function but were able to survive when receiving LPS challenged at 1mg/kg. However, greater fatality was observed when LPS dose was increased to 3 mg/kg. In 10-week-old (young adult) mice, 3 mg/kg LPS triggered heart dysfunction without impact on survival, whereases at 10 mg/kg, we observed significant LPS-induced fatality in the group. Due to the different sensitivities to LPS between the aged and young adult mice, we were not able to choose a universal dose of LPS to induce cardiac dysfunction and to perform follow up analysis in both groups. Therefore, we used the physiological function of the heart as a base for comparison in the studies performed in this report. Our previous research provided evidence that stimulating beclin-1 dependent autophagy improves cardiac performance during endotoxemia in young adult mice, and thus Beclin-1-activating peptide (TB peptide) holds a promising therapeutic potential for sepsis. In this report, we examined whether TB-peptide exerts a similar protective effect on aged animals under the same condition. In our experimental setting, sham or LPS challenge was administered to groups of 24-month-old and 10-week-old mice followed by treatment with TB-peptide, administered i.p. at a dose of 16 mg/kg in 100μl of PBS 30 minutes post LPS-challenge. Heart tissue was collected 18 hours post LPS challenge.

Sample Preparation:

Sampleprep ID:	SP002270
Sampleprep Summary:	Sample preparation: aqueous metabolites were extracted using a methanol-based protein precipitation method as described previously. Briefly, heart tissue samples were homogenized in cold water using zirconium oxide beads, methanol was added, and samples were vortexed and then stored for 30 minutes at -20˚C. Afterwards, samples were first sonicated in an ice bath for 10 minutes, centrifuged for 15 min at 18,000g and 4˚C, and then a fixed volume of supernatant was collected from each sample. Lastly, recovered supernatants were dried on a SpeedVac and reconstituted for LC-MS analysis. Protein pallets that were left over from the sample prep were saved for BCA assay. (2) LC-MS analysis: samples were analyzed on a duplex-LC-MS system composed of two Shimadzu UPLC pumps, CTC Analytics PAL HTC-xt temperature-controlled auto-sampler and AB Sciex 6500+ Triple Quadrupole MS equipped with ESI ionization source. UPLC pumps were connected to the auto-sampler in parallel and were able to perform two chromatography separations independently from each other. Each sample was injected twice on two identical analytical columns (Waters XBridge BEH Amide XP) performing separations in hydrophilic interaction liquid chromatography (HILIC) mode. While one column was performing separation and MS data acquisition in ESI(+) ionization mode, the other column was getting equilibrated for sample injection, chromatography separation and MS data acquisition in ESI(-) mode. Each chromatography separation was 18 minutes (total analysis time per sample was 36 minutes).

Sampleprep ID:

SP002270

Sampleprep Summary:

Sample preparation: aqueous metabolites were extracted using a methanol-based protein precipitation method as described previously. Briefly, heart tissue samples were homogenized in cold water using zirconium oxide beads, methanol was added, and samples were vortexed and then stored for 30 minutes at -20˚C. Afterwards, samples were first sonicated in an ice bath for 10 minutes, centrifuged for 15 min at 18,000g and 4˚C, and then a fixed volume of supernatant was collected from each sample. Lastly, recovered supernatants were dried on a SpeedVac and reconstituted for LC-MS analysis. Protein pallets that were left over from the sample prep were saved for BCA assay. (2) LC-MS analysis: samples were analyzed on a duplex-LC-MS system composed of two Shimadzu UPLC pumps, CTC Analytics PAL HTC-xt temperature-controlled auto-sampler and AB Sciex 6500+ Triple Quadrupole MS equipped with ESI ionization source. UPLC pumps were connected to the auto-sampler in parallel and were able to perform two chromatography separations independently from each other. Each sample was injected twice on two identical analytical columns (Waters XBridge BEH Amide XP) performing separations in hydrophilic interaction liquid chromatography (HILIC) mode. While one column was performing separation and MS data acquisition in ESI(+) ionization mode, the other column was getting equilibrated for sample injection, chromatography separation and MS data acquisition in ESI(-) mode. Each chromatography separation was 18 minutes (total analysis time per sample was 36 minutes).

Combined analysis:

Analysis ID	AN003566	AN003567
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Shimadzu Nexera X2	Shimadzu Nexera X2
Column	Waters XBridge BEH Amide (150 x 2.1mm,2.5um)	Waters XBridge BEH Amide (150 x 2.1mm,2.5um)
MS Type	ESI	ESI
MS instrument type	QTRAP	QTRAP
MS instrument name	ABI Sciex 6500+	ABI Sciex 6500+
Ion Mode	POSITIVE	NEGATIVE
Units	count per second (cps)	count per second (CPS)

Chromatography:

Chromatography ID:	CH002637
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters XBridge BEH Amide (150 x 2.1mm,2.5um)
Chromatography Type:	HILIC

Chromatography ID:	CH002638
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters XBridge BEH Amide (150 x 2.1mm,2.5um)
Chromatography Type:	HILIC

MS:

MS ID:	MS003323
Analysis ID:	AN003566
Instrument Name:	ABI Sciex 6500+
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	MS data acquisition was performed in multiple-reaction-monitoring (MRM) mode. The whole LC-MS system was controlled using AB Sciex Analyst 1.6.3 software. Measured MS peaks were integrated using AB Sciex MultiQuant 3.0.3 software. In addition to the study samples, two sets of quality control (QC) samples were used to monitor the assay performance as well as data reproducibility. One QC was a pooled human serum sample used to monitor system performance and the other QC was pooled study samples and this QC was used to monitor data reproducibility. Isotope labeled compounds were used to monitor sample preparation and injection. Highly reproducible MS data were generated, having an average coefficient of variances (CVs) among the metabolites of 5.6%. Data for each sample were normalized according to bicinchoninic acid (BCA)-based quantification of total protein count.
Ion Mode:	POSITIVE
Analysis Protocol File:	Zang_Protocol.docx

MS ID:	MS003324
Analysis ID:	AN003567
Instrument Name:	ABI Sciex 6500+
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	MS data acquisition was performed in multiple-reaction-monitoring (MRM) mode. The whole LC-MS system was controlled using AB Sciex Analyst 1.6.3 software. Measured MS peaks were integrated using AB Sciex MultiQuant 3.0.3 software. In addition to the study samples, two sets of quality control (QC) samples were used to monitor the assay performance as well as data reproducibility. One QC was a pooled human serum sample used to monitor system performance and the other QC was pooled study samples and this QC was used to monitor data reproducibility. Isotope labeled compounds were used to monitor sample preparation and injection. Highly reproducible MS data were generated, having an average coefficient of variances (CVs) among the metabolites of 5.6%. Data for each sample were normalized according to bicinchoninic acid (BCA)-based quantification of total protein count.
Ion Mode:	NEGATIVE
Analysis Protocol File:	Zang_Protocol.docx