#METABOLOMICS WORKBENCH cxs0651_20190131_075954 DATATRACK_ID:1614 STUDY_ID:ST001130 ANALYSIS_ID:AN001855 PROJECT_ID:PR000755 VERSION 1 CREATED_ON February 6, 2019, 9:36 am #PROJECT PR:PROJECT_TITLE Plasma metabolite profiling PR:PROJECT_TYPE MS untargeted data PR:PROJECT_SUMMARY Untargeted LC- data-dependent MS2 in positive mode to find difference in OIR PR:PROJECT_SUMMARY model mice and FG-4592 treated mice plasma. PR:INSTITUTE Cole eye institute, Cleveland Clinic PR:DEPARTMENT Cole eye institute PR:LABORATORY Dr. Jonathan Sears lab PR:LAST_NAME Singh PR:FIRST_NAME Charandeep PR:ADDRESS 9500 Euclid Avenue, Cleveland, Select One, 44195, USA PR:EMAIL cxs065@gmail.com PR:PHONE (216) 444-8232 #STUDY ST:STUDY_TITLE Urea cycle and 1C/serine metabolism in the prevention of oxygen induced ST:STUDY_TITLE retinopathy ST:STUDY_SUMMARY Untargeted metabolite profiling links the urea cycle and 1C/serine metabolism in ST:STUDY_SUMMARY the prevention of oxygen induced retinopathy by hepatic HIF stabilization. ST:STUDY_SUMMARY Premature infants require oxygen supplementation to survive that is ST:STUDY_SUMMARY simultaneously toxic to developing tissues. We have demonstrated that hypoxia ST:STUDY_SUMMARY inducible factor (HIF) stabilization during hyperoxia prevents oxygen induced ST:STUDY_SUMMARY retinopathy (OIR) and lung disease. Here, untargeted metabolite profiling ST:STUDY_SUMMARY coupled to XCMS systems biology analysis finds that serine/1C and urea cycles ST:STUDY_SUMMARY dominate pathway enrichment graphs. MS1 peak areas and MS2 library matches ST:STUDY_SUMMARY reveal 50% or more increased levels of plasma and retina serine, glycine, ST:STUDY_SUMMARY hypotaurine, methionine, and taurine. In addition, N-acetylglutamate increased ST:STUDY_SUMMARY 4-fold in serum, while orotate, citrulline, arginine, aspartate, glutamine were ST:STUDY_SUMMARY at least 50% increased after HIF stabilization. Targeted data analysis in vivo ST:STUDY_SUMMARY finds that retinal serine and glycine were derived from liver. HIF-1α2lox/2lox; ST:STUDY_SUMMARY albumin-cre KO had reduced levels of serine and retinal glycine. Inhibition of ST:STUDY_SUMMARY 1C metabolism blocked rescue by HIF stabilization. The metabolic phenotype of ST:STUDY_SUMMARY mice protected from OIR by HIF stabilization is dependent on hepatic serine/1C ST:STUDY_SUMMARY metabolism and urea cycle. ST:INSTITUTE Cole eye institute ST:LAST_NAME Singh ST:FIRST_NAME Charandeep ST:ADDRESS 9500 Euclid Avenue ST:EMAIL cxs065@gmail.com ST:PHONE (216) 444-8232 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Pos_FG1incluMS2 Treatment:Hyperoxia+FG4592 SUBJECT_SAMPLE_FACTORS - Pos_FG2incluMS2 Treatment:Hyperoxia+FG4593 SUBJECT_SAMPLE_FACTORS - Pos_FG3incluMS2 Treatment:Hyperoxia+FG4594 SUBJECT_SAMPLE_FACTORS - Pos_FG4incluMS2 Treatment:Hyperoxia+FG4595 SUBJECT_SAMPLE_FACTORS - Pos_FG5incluMS2 Treatment:Hyperoxia+FG4596 SUBJECT_SAMPLE_FACTORS - Pos_FG6incluMS2 Treatment:Hyperoxia+FG4597 SUBJECT_SAMPLE_FACTORS - Pos_hyper1incluMS2 Treatment:Hyperoxia SUBJECT_SAMPLE_FACTORS - Pos_hyper2incluMS2 Treatment:Hyperoxia SUBJECT_SAMPLE_FACTORS - Pos_hyper3incluMS2 Treatment:Hyperoxia SUBJECT_SAMPLE_FACTORS - Pos_hyper4incluMS2 Treatment:Hyperoxia SUBJECT_SAMPLE_FACTORS - Pos_hyper5incluMS2 Treatment:Hyperoxia SUBJECT_SAMPLE_FACTORS - Pos_hyper6incluMS2 Treatment:Hyperoxia #COLLECTION CO:COLLECTION_SUMMARY To extract the blood samples, mice were anesthetized with ketamine/xylazine mix CO:COLLECTION_SUMMARY and then blood was extracted from the heart of the animal with a 27.5 gauge CO:COLLECTION_SUMMARY needle fixed onto a 1cc heparinized syringe and added to 1.5 ml tubes containing CO:COLLECTION_SUMMARY 1.5 µl of Heparin 10,000 USP units/ml (Hospira, Lake forest, IL, USA) and CO:COLLECTION_SUMMARY stored on ice until further use. Plasma was obtained from blood samples by CO:COLLECTION_SUMMARY centrifuging samples at 1,100 x g at 4⁰C for 20 min. CO:SAMPLE_TYPE Blood (plasma) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY All experimental procedures involving live animals were conducted in accordance TR:TREATMENT_SUMMARY with the guidelines of the NIH Guide for the Care and Use of Laboratory Animals TR:TREATMENT_SUMMARY and approved by the Cleveland Clinic institutional animal care and use committee TR:TREATMENT_SUMMARY (IACUC, protocol #2016-1677). Wild type C57BL/6J mice were supplied by the TR:TREATMENT_SUMMARY Jackson Laboratory (Bar Harbor, ME). OIR model was based on the previously TR:TREATMENT_SUMMARY described procedure developed by LE Smith(Smith et al, 1994) and was described TR:TREATMENT_SUMMARY in detail elsewhere(Hoppe et al, 2014b; Sears et al, 2008; Singh et al, 2018). TR:TREATMENT_SUMMARY Metabolomics experiment was performed on p10 old OIR model mice Smith LE, TR:TREATMENT_SUMMARY Wesolowski E, McLellan A, Kostyk SK, D'Amato R, Sullivan R, D'Amore PA (1994) TR:TREATMENT_SUMMARY Oxygen-induced retinopathy in the mouse. Investigative ophthalmology & visual TR:TREATMENT_SUMMARY science 35: 101-111 Sears JE, Hoppe G, Ebrahem Q, Anand-Apte B (2008) Prolyl TR:TREATMENT_SUMMARY hydroxylase inhibition during hyperoxia prevents oxygen-induced retinopathy. TR:TREATMENT_SUMMARY Proceedings of the National Academy of Sciences 105: 19898-19903 Singh C, Sharma TR:TREATMENT_SUMMARY A, Hoppe G, Song W, Bolok Y, Sears JE (2018) 3-Hydroxypyruvate Destabilizes TR:TREATMENT_SUMMARY Hypoxia Inducible Factor and Induces Angiostasis. Investigative Ophthalmology & TR:TREATMENT_SUMMARY Visual Science 59: 3440-3448 #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites from plasma were extracted by adding 20 µl of plasma to 500 µl of SP:SAMPLEPREP_SUMMARY 50% cold acetonitrile. Samples were centrifuged at 15,000 x g for 10 min and 400 SP:SAMPLEPREP_SUMMARY µl supernatant was carefully removed into fresh tubes without disturbing the SP:SAMPLEPREP_SUMMARY cell pellet. Supernatants were injected directly into the LC-MS #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separation was performed on SeQuant ZIC-HILIC column with CH:CHROMATOGRAPHY_SUMMARY dimensions 150 x 2.1 mm, 3.5 µm, 100 Å (Merck, Darmstadt, Germany) attached to CH:CHROMATOGRAPHY_SUMMARY a precolumn SeQuant ZIC-HILIC with dimensions 20 x 2.1 mm, 3.5 µm, 100 Å CH:CHROMATOGRAPHY_SUMMARY (Merck, Darmstadt, Germany). The LC method used for separation of metabolites CH:CHROMATOGRAPHY_SUMMARY using the ZIC-HILIC column was adapted from Singh et al. al.(Singh et al, 2017). CH:CHROMATOGRAPHY_SUMMARY Briefly, gradient of solvent A (0.1% formic acid in water) and solvent B (0.08% CH:CHROMATOGRAPHY_SUMMARY formic acid in ACN) ramped from 80% B to 35% B in 23 minutes, followed by a wash CH:CHROMATOGRAPHY_SUMMARY step with 5% B from 25-30 min and then re-equilibration with 80% B from 25-30 CH:CHROMATOGRAPHY_SUMMARY min. Column oven was set to 20⁰C and a constant flow of solvents was set to CH:CHROMATOGRAPHY_SUMMARY 150 µl min-1. Samples were kept on 4⁰C auto-sampler throughout the CH:CHROMATOGRAPHY_SUMMARY measurements and 10 μl (plasma) of sample was injected. Singh C, Glaab E, CH:CHROMATOGRAPHY_SUMMARY Linster CL (2017) Molecular Identification of d-Ribulokinase in Budding Yeast CH:CHROMATOGRAPHY_SUMMARY and Mammals. The Journal of biological chemistry 292: 1005-1028 CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS LC-MS data from ddMS2 measurement were converted to mzML format using compound MS:MS_COMMENTS discoverer (version 2.1) (Thermo Scientific, Bellefonte, PA, USA) Data were MS:MS_COMMENTS analyzed using XCMS online tool(Forsberg et al, 2018). The metabolites were MS:MS_COMMENTS mapped onto the mouse database available on XCMS(Forsberg et al, 2018). Data was MS:MS_COMMENTS also analyzed with MSdial tool to confirm some of the metabolite annotations, MS:MS_COMMENTS using library provided with the MSdial software(Lai et al, 2017; Tsugawa et al, MS:MS_COMMENTS 2015). Forsberg EM, Huan T, Rinehart D, Benton HP, Warth B, Hilmers B, Siuzdak G MS:MS_COMMENTS (2018) Data processing, multi-omic pathway mapping, and metabolite activity MS:MS_COMMENTS analysis using XCMS Online. Nat Protoc 13: 633-651 Lai Z, Tsugawa H, Wohlgemuth MS:MS_COMMENTS G, Mehta S, Mueller M, Zheng Y, Ogiwara A, Meissen J, Showalter M, Takeuchi K, MS:MS_COMMENTS Kind T, Beal P, Arita M, Fiehn O (2017) Identifying metabolites by integrating MS:MS_COMMENTS metabolome databases with mass spectrometry cheminformatics. Nature methods 15: MS:MS_COMMENTS 53 Tsugawa H, Cajka T, Kind T, Ma Y, Higgins B, Ikeda K, Kanazawa M, MS:MS_COMMENTS VanderGheynst J, Fiehn O, Arita M (2015) MS-DIAL: data-independent MS/MS MS:MS_COMMENTS deconvolution for comprehensive metabolome analysis. Nature methods 12: 523-526 MS:MS_RESULTS_FILE ST001130_AN001855_Results.txt UNITS:Area under the curve Arbitrary units Has m/z:Yes Has RT:Yes RT units:Minutes #END