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MB Sample ID: SA091304

Local Sample ID:McN-Mat-20180411-01
Subject ID:SU001324
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:DBA/2J-mdx
Age Or Age Range:6 months
Gender:Male

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Subject:

Subject ID:SU001324
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:DBA/2J-mdx
Age Or Age Range:6 months
Gender:Male

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
McN-Mat-20180411-01SA091304FL013148vehicleTreatment

Collection:

Collection ID:CO001318
Collection Summary:3 vehicle control samples; 3 once-daily prednisone (1mg/kg i.p.) samples; 3 once-weekly prednisone (1mg/kg i.p.) samples. Each sample was extracted from ~100mg quadriceps muscle mass (one per mouse). Total hydrophilic metabolite content was extracted from quadriceps muscle tissue at treatment endpoint through methanol:water (80:20) extraction, adapting conditions described previously (Bruno et al., 2018). Briefly, total metabolite content from quadriceps muscle was obtained from ~100mg (wet weight) quadriceps muscle tissue per animal. Frozen (-80°C) muscle was pulverized in liquid nitrogen and homogenized with ~250l 2.3mm zirconia/silica beads (Cat # 11079125z, BioSpec, Bartlesville, OK) in 1ml methanol/water 80:20 (vol/vol) by means of Mini-BeadBeater-16 (Cat # 607, Biospec, Bartlesville, OK) for 1 minute. After centrifuging at 5000rpm for 5 minutes, 200l of supernatant were transferred into a tube pre-added with 800L of ice-cold methanol/water 80% (vol/vol). Samples were vortexed for 1 min, and then centrifuged at ~20,160 xg for 15 min at 4°C. Metabolite-containing extraction solution was then dried using SpeedVac (medium power).
Sample Type:Muscle

Treatment:

Treatment ID:TR001339
Treatment Summary:3 vehicle control samples; 3 once-daily prednisone (1mg/kg i.p.) samples; 3 once-weekly prednisone (1mg/kg i.p.) samples. Each sample was extracted from ~100mg quadriceps muscle mass (one per mouse). Treatment duration was four weeks. Samples were collected 48 hours after last prednisone injection.

Sample Preparation:

Sampleprep ID:SP001332
Sampleprep Summary:Total hydrophilic metabolite content was extracted from quadriceps muscle tissue at treatment endpoint through methanol:water (80:20) extraction, adapting conditions described previously (Bruno et al., 2018). Briefly, total metabolite content from quadriceps muscle was obtained from ~100mg (wet weight) quadriceps muscle tissue per animal. Frozen (-80°C) muscle was pulverized in liquid nitrogen and homogenized with ~250l 2.3mm zirconia/silica beads (Cat # 11079125z, BioSpec, Bartlesville, OK) in 1ml methanol/water 80:20 (vol/vol) by means of Mini-BeadBeater-16 (Cat # 607, Biospec, Bartlesville, OK) for 1 minute. After centrifuging at 5000rpm for 5 minutes, 200l of supernatant were transferred into a tube pre-added with 800L of ice-cold methanol/water 80% (vol/vol). Samples were vortexed for 1 min, and then centrifuged at ~20,160 xg for 15 min at 4°C. Metabolite-containing extraction solution was then dried using SpeedVac (medium power).

Combined analysis:

Analysis ID AN002085
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Ultimate3000
Column Waters XBridge Amide (100 x 4.6mm,3.5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q-Exactive
Ion Mode UNSPECIFIED
Units peak area values

Chromatography:

Chromatography ID:CH001522
Chromatography Summary:200ul of 50% Acetonitrile were added to the tube for reconstitution following by overtaxing for 1 min. Samples solution were then centrifuged for 15 min @ 20,000g, 4°C. Supernatant was collected for LC-MS analysis for Hydrophilic Metabolites Profiling as follows. Samples were analyzed by High-Performance Liquid Chromatography and High-Resolution Mass Spectrometry and Tandem Mass Spectrometry (HPLC-MS/MS). Specifically, system consisted of a Thermo Q-Exactive in line with an electrospray source and an Ultimate3000 (Thermo) series HPLC consisting of a binary pump, degasser, and auto-sampler outfitted with a Xbridge Amide column (Waters; dimensions of 4.6 mm × 100 mm and a 3.5 µm particle size). The mobile phase A contained 95% (vol/vol) water, 5% (vol/vol) acetonitrile, 20 mM ammonium hydroxide, 20 mM ammonium acetate, pH = 9.0; B was 100% Acetonitrile. The gradient was as following: 0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1-18 min, 75% A; 18-25 min, 15% A with a flow rate of 400 μL/min.
Instrument Name:Thermo Ultimate3000
Column Name:Waters XBridge Amide (100 x 4.6mm,3.5um)
Flow Gradient:0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1-18 min, 75% A; 18-25 min, 15% A
Flow Rate:400 µL/min
Solvent A: 95% water/5% acetonitrile; 20 mM ammonium hydroxide; 20 mM ammonium acetate, pH 9.0
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS001936
Analysis ID:AN002085
Instrument Name:Thermo Q-Exactive
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The capillary of the ESI source was set to 275 °C, with sheath gas at 45 arbitrary units, auxiliary gas at 5 arbitrary units and the spray voltage at 4.0 kV. In positive/negative polarity switching mode, an m/z scan range from 70 to 850 was chosen and MS1 data was collected at a resolution of 70,000. The automatic gain control (AGC) target was set at 1 × 106 and the maximum injection time was 200 ms. The top 5 precursor ions were subsequently fragmented, in a data-dependent manner, using the higher energy collisional dissociation (HCD) cell set to 30% normalized collision energy in MS2 at a resolution power of 17,500. The sample volumes of 25 μl were injected. Data acquisition and analysis were carried out by Xcalibur 4.0 software and Tracefinder 2.1 software, respectively (both from Thermo Fisher Scientific).
Ion Mode:UNSPECIFIED
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