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MB Sample ID: SA078091

Local Sample ID:Pos_FG3incluMS2
Subject ID:SU001191
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J

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Subject:

Subject ID:SU001191
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
Pos_FG3incluMS2SA078091FL011899Hyperoxia+FG4594Treatment

Collection:

Collection ID:CO001185
Collection Summary:To extract the blood samples, mice were anesthetized with ketamine/xylazine mix and then blood was extracted from the heart of the animal with a 27.5 gauge needle fixed onto a 1cc heparinized syringe and added to 1.5 ml tubes containing 1.5 µl of Heparin 10,000 USP units/ml (Hospira, Lake forest, IL, USA) and stored on ice until further use. Plasma was obtained from blood samples by centrifuging samples at 1,100 x g at 4⁰C for 20 min.
Sample Type:Blood (plasma)
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001206
Treatment Summary:All experimental procedures involving live animals were conducted in accordance with the guidelines of the NIH Guide for the Care and Use of Laboratory Animals and approved by the Cleveland Clinic institutional animal care and use committee (IACUC, protocol #2016-1677). Wild type C57BL/6J mice were supplied by the Jackson Laboratory (Bar Harbor, ME). OIR model was based on the previously described procedure developed by LE Smith(Smith et al, 1994) and was described in detail elsewhere(Hoppe et al, 2014b; Sears et al, 2008; Singh et al, 2018). Metabolomics experiment was performed on p10 old OIR model mice Smith LE, Wesolowski E, McLellan A, Kostyk SK, D'Amato R, Sullivan R, D'Amore PA (1994) Oxygen-induced retinopathy in the mouse. Investigative ophthalmology & visual science 35: 101-111 Sears JE, Hoppe G, Ebrahem Q, Anand-Apte B (2008) Prolyl hydroxylase inhibition during hyperoxia prevents oxygen-induced retinopathy. Proceedings of the National Academy of Sciences 105: 19898-19903 Singh C, Sharma A, Hoppe G, Song W, Bolok Y, Sears JE (2018) 3-Hydroxypyruvate Destabilizes Hypoxia Inducible Factor and Induces Angiostasis. Investigative Ophthalmology & Visual Science 59: 3440-3448

Sample Preparation:

Sampleprep ID:SP001199
Sampleprep Summary:Metabolites from plasma were extracted by adding 20 µl of plasma to 500 µl of 50% cold acetonitrile. Samples were centrifuged at 15,000 x g for 10 min and 400 µl supernatant was carefully removed into fresh tubes without disturbing the cell pellet. Supernatants were injected directly into the LC-MS

Combined analysis:

Analysis ID AN001855
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Vanquish
Column SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE
Units Area under the curve Arbitrary units

Chromatography:

Chromatography ID:CH001342
Chromatography Summary:Chromatographic separation was performed on SeQuant ZIC-HILIC column with dimensions 150 x 2.1 mm, 3.5 µm, 100 Å (Merck, Darmstadt, Germany) attached to a precolumn SeQuant ZIC-HILIC with dimensions 20 x 2.1 mm, 3.5 µm, 100 Å (Merck, Darmstadt, Germany). The LC method used for separation of metabolites using the ZIC-HILIC column was adapted from Singh et al. al.(Singh et al, 2017). Briefly, gradient of solvent A (0.1% formic acid in water) and solvent B (0.08% formic acid in ACN) ramped from 80% B to 35% B in 23 minutes, followed by a wash step with 5% B from 25-30 min and then re-equilibration with 80% B from 25-30 min. Column oven was set to 20⁰C and a constant flow of solvents was set to 150 µl min-1. Samples were kept on 4⁰C auto-sampler throughout the measurements and 10 μl (plasma) of sample was injected. Singh C, Glaab E, Linster CL (2017) Molecular Identification of d-Ribulokinase in Budding Yeast and Mammals. The Journal of biological chemistry 292: 1005-1028
Instrument Name:Thermo Vanquish
Column Name:SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
Column Temperature:20
Flow Gradient:ramped from 80% B to 35% B in 23 minutes, followed by a wash step with 5% B from 25-30 min and then re-equilibration with 80% B from 25-30 min.
Flow Rate:150 µl/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.08% formic acid
Chromatography Type:HILIC

MS:

MS ID:MS001715
Analysis ID:AN001855
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:LC-MS data from ddMS2 measurement were converted to mzML format using compound discoverer (version 2.1) (Thermo Scientific, Bellefonte, PA, USA) Data were analyzed using XCMS online tool(Forsberg et al, 2018). The metabolites were mapped onto the mouse database available on XCMS(Forsberg et al, 2018). Data was also analyzed with MSdial tool to confirm some of the metabolite annotations, using library provided with the MSdial software(Lai et al, 2017; Tsugawa et al, 2015). Forsberg EM, Huan T, Rinehart D, Benton HP, Warth B, Hilmers B, Siuzdak G (2018) Data processing, multi-omic pathway mapping, and metabolite activity analysis using XCMS Online. Nat Protoc 13: 633-651 Lai Z, Tsugawa H, Wohlgemuth G, Mehta S, Mueller M, Zheng Y, Ogiwara A, Meissen J, Showalter M, Takeuchi K, Kind T, Beal P, Arita M, Fiehn O (2017) Identifying metabolites by integrating metabolome databases with mass spectrometry cheminformatics. Nature methods 15: 53 Tsugawa H, Cajka T, Kind T, Ma Y, Higgins B, Ikeda K, Kanazawa M, VanderGheynst J, Fiehn O, Arita M (2015) MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nature methods 12: 523-526
Ion Mode:POSITIVE
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