Summary of Study ST002830

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001772. The data can be accessed directly via it's Project DOI: 10.21228/M8NT5Z This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002830
Study Title	L-isoleucine in P10 STZ
Study Summary	Summary Reactive oxygen species (ROS) are by-products of metabolism of oxygen and they play an important role in normal homeostasis and cell signaling, as well as in the initiation of diseases including cancer when their production is upregulated. Thus, it is imperative to understand the cellular and molecular basis by which ROS impact on various biological and pathological processes. Here, we identified 2-oxindole, a tryptophan derivative, was a major catabolic product in hydrogen peroxide-treated cell culture medium. We used 2-oxindole to study its role in regulating AhR signaling and tryptophan metabolic pathways. We found that 2-oxindole significantly increased the activity of AhR, leading to enhanced expression of its downstream targets including cytochrome P450 genes.
Institute	Boston Children's Hospital
Last Name	Fu
First Name	Zhongjie
Address	1 Blackfan Circle, Boston, MA 02114
Email	Zhongjie.Fu@childrens.harvard.edu
Phone	6179192534
Submit Date	2023-08-10
Num Groups	2
Total Subjects	6
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2023-09-14
Release Version	1

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Project:

Project ID:	PR001772
Project DOI:	doi: 10.21228/M8NT5Z
Project Title:	L-isoleucine in P10 STZ (Streptozotocin)
Project Summary:	The effects of intraperitoneal L-isoleucine injection in murine hyperglycemia-associated retinopathy, a model mimicking aspects of retinopathy of prematurity (ROP), will be studied. ROP is a leading cause of blindness in children worldwide. Amino acid metabolism is altered in preterm infants. In this study, pups were daily injected with streptozotocin from P1 to P10 to induce hyperglycemia-associated retinopathy. L-isoleucine or PBS control was injected intraperitoneally from P7 to P10. Retinas were collected at P10. 8 retinas (from 4 pups) were pooled to generate 1 sample. 3 samples per group will be compared.
Institute:	Boston Childrens Hospital
Last Name:	Fu
First Name:	Zhongjie
Address:	1 Blackfan Circle, Boston, MA 02114
Email:	Zhongjie.Fu@childrens.harvard.edu
Phone:	617-919-2534

Subject:

Subject ID:	SU002939
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Group
SA306482	Blank_2	Blank
SA306483	Blank_3	Blank
SA306484	Blank_5	Blank
SA306485	Blank_0	Blank
SA306486	Blank_4	Blank
SA306487	Blank_1	Blank
SA306488	ISTD_2	ISTD
SA306489	ISTD_0	ISTD
SA306490	ISTD_1	ISTD
SA306491	L-isoleucine 2	L-isoleucine
SA306492	L-isoleucine 3	L-isoleucine
SA306493	L-isoleucine 1	L-isoleucine
SA306494	PBS 3	PBS
SA306495	PBS 1	PBS
SA306496	PBS 2	PBS

Showing results 1 to 15 of 15

Showing results 1 to 15 of 15

Collection:

Collection ID:	CO002932
Collection Summary:	Retinas were collected at P10 following a single incision across the sclera and immediately snap frozen in liquid nitrogen and stored at -80 ºC until sample processing 2. Samples were processed and analyzed by LC-MS/MS by the NYU Metabolomics Core Resource Laboratory, New York, NY, USA, as described previously 3,4. Briefly, samples were homogenized using a bead blaster for 10 cycles with 30 seconds on and 30 seconds off. Metabolites were extracted using 80% methanol and dried down using a speedvac. Next, samples were reconstituted in 50 µL MS-grade water and sonicated for two minutes. Samples were then spun down in a centrifuge at 10 G for 4 min and finally transferred to MS vials for analysis. Internal standards were used for correction of retention time and identification of metabolites. Six retinas were pooled as one replicate to reduce biological variability for metabolomics analysis in each group, n=6 per group (HAR vs. control)
Sample Type:	Retina

Treatment:

Treatment ID:	TR002948
Treatment Summary:	To study the metabolic alterations occurring in hyperglycemia-associated Phase I ROP, we applied quantitative metabolomics and proteomics on mouse retinas from HAR and normal control mice. C57BL/6J (Jackson Laboratory, Bar Harbor, ME) mice, of each sex, aged 10-12 weeks, were purchased, housed and bred in the institutional vivarium and maintained on a 12hour/12hour light/dark cycle with mouse chow provided ad libitum. Neonatal mice were randomly assigned to experimental groups. Induction of hyperglycemia was accomplished as previously described 1. Neonatal mice were intraperitoneally injected with 50mg/kg/day STZ consecutively from P1 to P9 using a 34-G needle (Hamilton syringe) (Fig. 1B). Vehicle control animals received equal volumes of vehicle phosphate-buffered saline (PBS, Gibco, Waltham, MA). Hyperglycemia is induced around P8 and delayed retinal vascularization is found at P10 1. Mice with weight range 4 to 5 grams were used for further metabolomics and proteomics analysis. Mouse litters were randomly assigned to HAR or control groups, both sexes were used. The cages were located at close spots to minimize the potential housing influences. All procedures were approved by our Institutional Animal Care and Use Committee and adhered to ARRIVE guidelines and the NIH Guide for the Care and Use of Laboratory Animals. With conditions tested with β=0.8 and α=0.05, at least n=6 per group will be needed for the analysis. Control was re-named as group 1 and HAR was re-named as group 2 for analysis.

Treatment ID:

TR002948

Treatment Summary:

To study the metabolic alterations occurring in hyperglycemia-associated Phase I ROP, we applied quantitative metabolomics and proteomics on mouse retinas from HAR and normal control mice. C57BL/6J (Jackson Laboratory, Bar Harbor, ME) mice, of each sex, aged 10-12 weeks, were purchased, housed and bred in the institutional vivarium and maintained on a 12hour/12hour light/dark cycle with mouse chow provided ad libitum. Neonatal mice were randomly assigned to experimental groups. Induction of hyperglycemia was accomplished as previously described 1. Neonatal mice were intraperitoneally injected with 50mg/kg/day STZ consecutively from P1 to P9 using a 34-G needle (Hamilton syringe) (Fig. 1B). Vehicle control animals received equal volumes of vehicle phosphate-buffered saline (PBS, Gibco, Waltham, MA). Hyperglycemia is induced around P8 and delayed retinal vascularization is found at P10 1. Mice with weight range 4 to 5 grams were used for further metabolomics and proteomics analysis. Mouse litters were randomly assigned to HAR or control groups, both sexes were used. The cages were located at close spots to minimize the potential housing influences. All procedures were approved by our Institutional Animal Care and Use Committee and adhered to ARRIVE guidelines and the NIH Guide for the Care and Use of Laboratory Animals. With conditions tested with β=0.8 and α=0.05, at least n=6 per group will be needed for the analysis. Control was re-named as group 1 and HAR was re-named as group 2 for analysis.

Sample Preparation:

Sampleprep ID:	SP002945
Sampleprep Summary:	Both retinas from each mouse were collected, pooled, and prepared for targeted MS-based proteomics as described previously 1. After sample preparation, LC-tandem MS analysis was performed using selected reaction monitoring (SRM) 5 on a Thermo Scientific TSQ Vantage mass spectrometer equipped with an Eksigent splitless nanoflow HPLC system. 7 µL aliquots of each sample were injected onto a 10 cm x 75 µm i.d. capillary column packed with Phenomenex Jupiter C18 reversed phase beads. The column was eluted at 150 nL/min with a 60 min linear gradient of acetonitrile in 0.1% formic acid. The SRM assays were developed and validated to monitor two peptides per protein. Each peptide was monitored in a 6-min window centered on the known elution time of the peptide 6-8. Approximately 30 protein assays are grouped into a panel of proteins that are measured in a single LC-tandem MS run.
Sampleprep Protocol ID:	SP002600

Combined analysis:

Analysis ID	AN004623
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Ultimate 3000
Column	SeQuant ZIC-pHILIC (150 x 2.1mm, 5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED
Units	abundance/intensity

Chromatography:

Chromatography ID:	CH003479
Instrument Name:	Thermo Ultimate 3000
Column Name:	SeQuant ZIC-pHILIC (150 x 2.1mm, 5um)
Column Temperature:	25
Flow Gradient:	80-20%B (0-30 min), 20-80%B (30-31 min), 80-80%B (31-42 min)
Flow Rate:	0.1mL/min
Solvent A:	100% water; 10 mM ammonium carbonate, pH 9.0
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS004369
Analysis ID:	AN004623
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Xcalibur, chromeleon unspecified because both negative and positive ion mode were collected
Ion Mode:	UNSPECIFIED