#METABOLOMICS WORKBENCH juliehaines_20211122_115205 DATATRACK_ID:2938 STUDY_ID:ST001997 ANALYSIS_ID:AN003258 PROJECT_ID:000000 VERSION 1 CREATED_ON November 22, 2021, 12:06 pm #PROJECT PR:PROJECT_TITLE Polyamine import and accumulation causes immunomodulation in macrophages PR:PROJECT_TITLE engulfing apoptotic cells PR:PROJECT_SUMMARY Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue PR:PROJECT_SUMMARY homeostasis and drives anti-inflammatory programming in engulfing macrophages. PR:PROJECT_SUMMARY Here, we assess metabolites in naïve and inflammatory macrophages following PR:PROJECT_SUMMARY engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to PR:PROJECT_SUMMARY unique increases in the arginine-derived polyamines, spermidine and spermine, in PR:PROJECT_SUMMARY vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does PR:PROJECT_SUMMARY not arise from retention of apoptotic cell metabolites or de novo synthesis, but PR:PROJECT_SUMMARY from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. PR:PROJECT_SUMMARY Blocking polyamine import prevents efferocytosis from suppressing macrophage PR:PROJECT_SUMMARY IL-1or IL-6. This identifies efferocytosis as a trigger for polyamine PR:PROJECT_SUMMARY import and accumulation, and imported polyamines as mediators of PR:PROJECT_SUMMARY efferocytosis-induced immune reprogramming. PR:INSTITUTE University of Colorado Denver PR:LAST_NAME Haines PR:FIRST_NAME Julie PR:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA PR:EMAIL julie.haines@cuanschutz.edu PR:PHONE 3037243339 #STUDY ST:STUDY_TITLE Polyamine import and accumulation causes immunomodulation in macrophages ST:STUDY_TITLE engulfing apoptotic cells (Part 2) ST:STUDY_SUMMARY Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue ST:STUDY_SUMMARY homeostasis and drives anti-inflammatory programming in engulfing macrophages. ST:STUDY_SUMMARY Here, we assess metabolites in naïve and inflammatory macrophages following ST:STUDY_SUMMARY engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to ST:STUDY_SUMMARY unique increases in the arginine-derived polyamines, spermidine and spermine, in ST:STUDY_SUMMARY vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does ST:STUDY_SUMMARY not arise from retention of apoptotic cell metabolites or de novo synthesis, but ST:STUDY_SUMMARY from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. ST:STUDY_SUMMARY Blocking polyamine import prevents efferocytosis from suppressing macrophage ST:STUDY_SUMMARY IL-1or IL-6. This identifies efferocytosis as a trigger for polyamine ST:STUDY_SUMMARY import and accumulation, and imported polyamines as mediators of ST:STUDY_SUMMARY efferocytosis-induced immune reprogramming. ST:INSTITUTE University of Colorado Denver ST:LAST_NAME Haines ST:FIRST_NAME Julie ST:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA ST:EMAIL julie.haines@cuanschutz.edu ST:PHONE 3037243339 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - 4h 1 category:4h unexposed RAW_FILE_NAME=37 SUBJECT_SAMPLE_FACTORS - 4h 2 category:4h unexposed RAW_FILE_NAME=40 SUBJECT_SAMPLE_FACTORS - 4h 3 category:4h unexposed RAW_FILE_NAME=43 SUBJECT_SAMPLE_FACTORS - 4h 1- category:4h non-engulfing RAW_FILE_NAME=38 SUBJECT_SAMPLE_FACTORS - 4h 2- category:4h non-engulfing RAW_FILE_NAME=41 SUBJECT_SAMPLE_FACTORS - 4h 3- category:4h non-engulfing RAW_FILE_NAME=44 SUBJECT_SAMPLE_FACTORS - 4h 1+ category:4h engulfing RAW_FILE_NAME=39 SUBJECT_SAMPLE_FACTORS - 4h 2+ category:4h engulfing RAW_FILE_NAME=42 SUBJECT_SAMPLE_FACTORS - 4h 3+ category:4h engulfing RAW_FILE_NAME=45 SUBJECT_SAMPLE_FACTORS - 8h 1 category:8h unexposed RAW_FILE_NAME=46 SUBJECT_SAMPLE_FACTORS - 8h 2 category:8h unexposed RAW_FILE_NAME=49 SUBJECT_SAMPLE_FACTORS - 8h 3 category:8h unexposed RAW_FILE_NAME=52 SUBJECT_SAMPLE_FACTORS - 8h 1- category:8h non-engulfing RAW_FILE_NAME=47 SUBJECT_SAMPLE_FACTORS - 8h 2- category:8h non-engulfing RAW_FILE_NAME=50 SUBJECT_SAMPLE_FACTORS - 8h 3- category:8h non-engulfing RAW_FILE_NAME=53 SUBJECT_SAMPLE_FACTORS - 8h 1+ category:8h engulfing RAW_FILE_NAME=48 SUBJECT_SAMPLE_FACTORS - 8h 2+ category:8h engulfing RAW_FILE_NAME=51 SUBJECT_SAMPLE_FACTORS - 8h 3+ category:8h engulfing RAW_FILE_NAME=54 #COLLECTION CO:COLLECTION_SUMMARY Experimental animals This study was approved and performed in accordance with CO:COLLECTION_SUMMARY the ethical guidelines of the Institutional Animal Care and Use Committee at CO:COLLECTION_SUMMARY National Jewish Health. C57BL/6J (000664), B6-CD45.1 (002014) and CCR2KO CO:COLLECTION_SUMMARY (004999) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). CO:COLLECTION_SUMMARY DsRed.T3 mice (available as 006051 at Jackson Labs) were generously shared by CO:COLLECTION_SUMMARY the Nagy laboratory (Vintersten et al., 2004). CCR2KO mice were crossed with CO:COLLECTION_SUMMARY DsRed mice until double homozygous. Mice used in experiments were between 8-16 CO:COLLECTION_SUMMARY weeks of age. Both male and female sex were used in experiments except bone CO:COLLECTION_SUMMARY marrow chimera experiments, which used only male mice. Human tissue This study CO:COLLECTION_SUMMARY used deidentified donor peripheral blood mononuclear cells (PBMC) collected and CO:COLLECTION_SUMMARY isolated (Young et al., 2011) by the National Jewish Blood Prep Core (IRB No. CO:COLLECTION_SUMMARY HS-1285). Age and sex of deidentified blood donors in this study is not known. CO:COLLECTION_SUMMARY Cell Lines Jurkat (human T lymphocytes, clone E6-1) were obtained from ATCC and CO:COLLECTION_SUMMARY cultured in RPMI-1640 (GIBCO) supplemented with 10% heat-inactivated fetal CO:COLLECTION_SUMMARY bovine serum (v/v) (GeminiBio), 1X Pen/Strep/Glut, 1mM HEPES, and 100M Sodium CO:COLLECTION_SUMMARY Pyruvate. Cells were cultured in a humidified CO2 incubator at 37C. The Jurkat CO:COLLECTION_SUMMARY cell line was established from peripheral blood of a 14-year-old male diagnosed CO:COLLECTION_SUMMARY with T cell leukemia. Murine primary cell cultures Peritoneal cells were CO:COLLECTION_SUMMARY isolated from naïve mice by lavage with 10mL of PBS containing 0.5mM EDTA. CO:COLLECTION_SUMMARY Lavage was estimated to contain 50% macrophages. Peritoneal macrophages were CO:COLLECTION_SUMMARY isolated by adhesion purification of lavage cells in RPMI-10 (RPMI-1640 CO:COLLECTION_SUMMARY containing 10% heat-inactivated fetal bovine serum (v/v), 1X Pen/Strep/Glut, 1mM CO:COLLECTION_SUMMARY HEPES, and 100M Sodium Pyruvate) plated 1.3x106 macrophages/well in 6-well CO:COLLECTION_SUMMARY plates for FACS sorting or 4x106 macrophage/well in 24-well plates for flow CO:COLLECTION_SUMMARY cytometry. Non-adherent cells were removed by washing after 3-5 hours, and CO:COLLECTION_SUMMARY macrophages were cultured in fresh RPMI-10 overnight. 400ng/mL purified E. coli CO:COLLECTION_SUMMARY O111:B4 LPS (List Biological Laboratories Inc.) was added to generate CO:COLLECTION_SUMMARY inflammatory macrophages 12h before the addition of target cells. Bone CO:COLLECTION_SUMMARY marrow-derived macrophages were generated by 8-day culture of murine bone marrow CO:COLLECTION_SUMMARY cells grown in media containing M-CSF (High glucose DMEM containing 10% v/v FBS, CO:COLLECTION_SUMMARY 20% v/v L929-conditioned media, 1X Pen/Strep/Glut, 100M Sodium Pyruvate, CO:COLLECTION_SUMMARY 55M beta-Mercaptoethanol). All macrophages were removed from culture dishes CO:COLLECTION_SUMMARY by 3-minute incubation with 1X Trypsin-EDTA (Sigma) plus gentle scraping. CO:SAMPLE_TYPE Macrophages #TREATMENT TR:TREATMENT_SUMMARY LPS-primed murine peritoneal macrophages were fed fluorescently-tagged TR:TREATMENT_SUMMARY IgG-opsonized live Jurkat cells for 1h. Unengulfed targets were washed off and TR:TREATMENT_SUMMARY macrophages were left to degrade targets for a further 3 or 7 hours. Macrophages TR:TREATMENT_SUMMARY were then collected and sorted into non-engulfing or engulfing populations, TR:TREATMENT_SUMMARY alongside control unexposed macrophages. Sorted cells were pelleted at 400xg and TR:TREATMENT_SUMMARY dry pellets were snap frozen and stored at -80C until metabolite extraction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To process cells for assessment of intracellular metabolites, cells were SP:SAMPLEPREP_SUMMARY pelleted at 400xg in tubes coated with 0.06% BSA. Supernatant was aspirated and SP:SAMPLEPREP_SUMMARY discarded; residual liquid was carefully wicked away from the pellet with a SP:SAMPLEPREP_SUMMARY kimwipe. Dry pellets were immediately snap frozen and stored at -80C until SP:SAMPLEPREP_SUMMARY processing. To process culture supernatants for assessment of metabolites, SP:SAMPLEPREP_SUMMARY supernatant was centrifuged at 400xg to pellet any cells. Cell-free supernatant SP:SAMPLEPREP_SUMMARY was then transferred to a fresh tube, snap frozen, and stored at -80C until SP:SAMPLEPREP_SUMMARY processing. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Isocratic flow of 100% phase A (95% water, 5% acetonitrile, 10 mM ammonium CH:CHROMATOGRAPHY_SUMMARY acetate) at 250 uL/min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um) CH:FLOW_GRADIENT isocratic CH:FLOW_RATE 250 ul/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Global metabolomics analyses were performed using a 3 min isocratic run in MS:MS_COMMENTS positive and negative ion modes (separate runs) as described previously (Nemkov MS:MS_COMMENTS et al., 2015, Nemkov et al., 2017); stable isotope tracing samples were analyzed MS:MS_COMMENTS using a 5 min C18 gradient in positive and negative ion modes (separate runs) as MS:MS_COMMENTS described (Nemkov et al., 2019, Gehrke et al., 2019). For all analyses, the MS MS:MS_COMMENTS scanned in MS1 mode across the m/z range of 65 to 950. Peaks were annotated (in MS:MS_COMMENTS conjunction with the KEGG database), integrated, and quality control performed MS:MS_COMMENTS using Maven (Princeton University) as described. Stable isotope tracing results MS:MS_COMMENTS were isotopically corrected for the natural abundance of 13C1, 13C2, 15N1, and MS:MS_COMMENTS 15N2 (Nemkov et al., 2017). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples 4h 1 4h 2 4h 3 4h 1- 4h 2- 4h 3- 4h 1+ 4h 2+ 4h 3+ 8h 1 8h 2 8h 3 8h 1- 8h 2- 8h 3- 8h 1+ 8h 2+ 8h 3+ Factors category:4h unexposed category:4h unexposed category:4h unexposed category:4h non-engulfing category:4h non-engulfing category:4h non-engulfing category:4h engulfing category:4h engulfing category:4h engulfing category:8h unexposed category:8h unexposed category:8h unexposed category:8h non-engulfing category:8h non-engulfing category:8h non-engulfing category:8h engulfing category:8h engulfing category:8h engulfing ATP 1.46E+04 1.37E+05 6.61E+04 4.26E+04 5.42E+04 1.00E+05 2.49E+05 1.23E+05 2.14E+04 1.17E+04 1.10E+05 2.84E+05 2.06E+05 1.87E+05 1.94E+05 4.83E+05 3.20E+04 2.50E+05 UTP 4.37E+04 9.11E+04 4.92E+04 1.46E+04 6.39E+04 9.77E+04 9.00E+04 7.77E+04 4.51E+04 4.07E+04 6.24E+04 9.99E+04 6.55E+04 6.71E+04 7.48E+04 1.10E+05 5.74E+04 9.72E+04 UDP 5.48E+04 2.79E+05 1.97E+05 6.44E+04 2.79E+05 2.21E+05 2.35E+05 2.68E+05 6.71E+04 5.46E+04 1.85E+05 2.27E+05 2.51E+05 1.62E+05 2.21E+05 2.55E+05 5.47E+04 1.76E+05 IDP 1.95E+06 3.09E+06 3.08E+06 3.44E+06 2.87E+06 2.26E+06 2.42E+06 3.21E+06 2.67E+06 2.34E+06 2.62E+06 2.31E+06 2.40E+06 2.58E+06 2.60E+06 2.43E+06 2.56E+06 2.40E+06 Pyridoxal 6.95E+04 7.53E+04 5.61E+04 5.87E+04 8.11E+04 6.39E+04 7.31E+04 7.10E+04 6.94E+04 6.41E+04 9.29E+04 7.68E+04 7.99E+04 8.69E+04 7.62E+04 9.88E+04 6.39E+04 7.87E+04 UDP-glucose 4.87E+04 3.37E+04 3.19E+04 3.78E+04 2.69E+04 5.54E+04 5.93E+04 5.30E+04 4.14E+04 4.41E+04 5.37E+04 4.86E+04 5.26E+04 6.10E+04 5.32E+04 6.86E+04 7.38E+04 7.14E+04 Phosphate 9.53E+07 1.10E+08 1.09E+08 1.06E+08 1.12E+08 1.08E+08 8.97E+07 8.38E+07 1.02E+08 1.00E+08 9.23E+07 1.11E+08 1.06E+08 1.06E+08 1.13E+08 9.98E+07 1.39E+08 1.12E+08 Diphosphate 6.24E+06 7.36E+06 7.27E+06 6.54E+06 6.21E+06 7.21E+06 5.08E+06 3.91E+06 7.52E+06 6.52E+06 6.39E+06 7.50E+06 6.98E+06 7.41E+06 8.00E+06 6.25E+06 1.32E+07 8.40E+06 D-Glucose 1.38E+07 1.76E+07 1.40E+07 1.49E+07 1.50E+07 1.28E+07 1.67E+07 1.76E+07 1.23E+07 1.17E+07 1.23E+07 1.45E+07 1.48E+07 1.25E+07 1.43E+07 1.74E+07 1.06E+07 1.22E+07 D-Glucose 6-phosphate 4.15E+05 4.30E+05 3.95E+05 4.72E+05 3.68E+05 5.92E+05 6.53E+05 5.01E+05 6.92E+05 3.26E+05 3.89E+05 3.90E+05 3.59E+05 3.59E+05 4.08E+05 4.68E+05 3.81E+05 5.53E+05 D-Fructose 1-6-bisphosphate 8.59E+05 1.56E+06 1.04E+06 6.75E+05 7.81E+05 1.32E+06 1.72E+06 1.48E+06 1.48E+06 6.40E+05 8.15E+05 8.60E+05 6.92E+05 8.34E+05 9.11E+05 8.36E+05 9.94E+05 1.20E+06 D-Glyceraldehyde 3-phosphate/Glycerone phosphate 9.57E+06 1.19E+06 3.35E+06 2.22E+06 2.54E+06 4.80E+06 7.98E+06 9.07E+06 2.91E+06 4.04E+06 4.37E+06 1.13E+06 2.82E+06 2.92E+06 6.21E+06 2.02E+06 3.33E+06 4.92E+06 2/3-Phospho-D-glycerate 3.18E+05 4.14E+05 3.07E+05 3.55E+05 3.28E+05 4.55E+05 3.84E+05 3.33E+05 3.58E+05 2.95E+05 3.46E+05 4.23E+05 4.08E+05 3.79E+05 4.36E+05 3.79E+05 3.87E+05 4.46E+05 Phosphoenolpyruvate 7.66E+04 1.45E+05 7.80E+04 9.82E+04 1.24E+05 1.79E+05 1.35E+05 7.48E+04 5.68E+04 8.52E+04 2.14E+05 9.52E+04 9.22E+04 9.08E+04 9.59E+04 1.13E+05 6.67E+04 7.78E+04 Pyruvate 5.73E+05 7.40E+05 5.72E+05 5.83E+05 6.46E+05 4.76E+05 7.05E+05 8.20E+05 6.11E+05 5.64E+05 5.70E+05 5.69E+05 6.95E+05 5.42E+05 7.08E+05 7.24E+05 3.76E+05 5.54E+05 Lactate 5.03E+06 4.64E+06 4.01E+06 4.87E+06 4.79E+06 4.08E+06 5.36E+06 5.92E+06 4.61E+06 3.86E+06 4.67E+06 4.69E+06 5.38E+06 4.30E+06 5.94E+06 6.33E+06 3.57E+06 5.03E+06 D-Ribose 3.55E+05 3.63E+05 3.83E+05 3.98E+05 3.59E+05 3.17E+05 3.96E+05 3.83E+05 2.76E+05 2.85E+05 3.46E+05 3.42E+05 3.30E+05 3.21E+05 3.38E+05 4.18E+05 2.80E+05 3.02E+05 D-Rhamnose 1.38E+06 1.51E+06 9.77E+05 1.42E+06 1.39E+06 1.39E+06 1.52E+06 9.70E+05 1.42E+06 1.07E+06 1.21E+06 1.37E+06 9.99E+05 1.18E+06 1.31E+06 1.35E+06 9.80E+05 1.20E+06 Citrate 3.46E+06 4.52E+06 3.74E+06 4.20E+06 3.64E+06 5.94E+06 7.22E+06 4.31E+06 5.68E+06 3.59E+06 3.95E+06 5.94E+06 5.24E+06 5.13E+06 6.99E+06 8.19E+06 6.10E+06 8.81E+06 2-Oxoglutarate 5.94E+04 7.45E+04 4.97E+04 5.24E+04 5.99E+04 6.89E+04 8.65E+04 8.72E+04 6.12E+04 8.52E+04 8.07E+04 5.68E+04 8.61E+04 6.90E+04 7.48E+04 1.13E+05 8.88E+04 6.52E+04 Succinate 4.86E+05 5.87E+05 5.20E+05 7.58E+05 5.18E+05 5.18E+05 6.31E+05 6.51E+05 5.14E+05 5.27E+05 6.51E+05 5.21E+05 8.44E+05 6.81E+05 5.96E+05 1.06E+06 7.58E+05 6.37E+05 Fumarate 7.61E+04 8.16E+04 5.05E+04 5.41E+04 6.59E+04 8.58E+04 1.27E+05 8.40E+04 1.28E+05 6.09E+04 6.42E+04 5.89E+04 7.18E+04 8.50E+04 9.50E+04 1.16E+05 1.15E+05 1.29E+05 Malate 7.44E+05 1.09E+06 7.85E+05 6.10E+05 6.40E+05 1.13E+06 1.44E+06 9.91E+05 1.85E+06 7.47E+05 9.27E+05 8.20E+05 9.01E+05 9.35E+05 1.34E+06 1.37E+06 1.52E+06 1.72E+06 Itaconate 4.72E+06 4.93E+06 3.44E+06 4.23E+06 3.56E+06 4.68E+06 1.00E+07 6.32E+06 5.74E+06 6.21E+06 5.87E+06 4.66E+06 7.97E+06 7.58E+06 5.51E+06 1.27E+07 1.07E+07 7.29E+06 2-Hydroxyglutarate/Citramalate 4.47E+05 5.09E+05 4.26E+05 4.46E+05 3.63E+05 6.08E+05 5.23E+05 3.44E+05 5.38E+05 4.07E+05 5.20E+05 4.32E+05 5.64E+05 5.46E+05 4.40E+05 6.26E+05 6.63E+05 5.05E+05 Sedoheptulose 1-phosphate 6.71E+04 8.51E+04 5.12E+04 5.88E+04 4.84E+04 9.16E+04 9.21E+04 7.73E+04 1.04E+05 4.34E+04 5.11E+04 4.94E+04 4.55E+04 7.17E+04 2.57E+04 1.00E+05 4.91E+04 9.69E+04 alpha-D-Ribose 1-phosphate 1.73E+05 1.98E+05 2.11E+05 1.86E+05 2.11E+05 1.49E+05 2.08E+05 1.94E+05 1.65E+05 1.40E+05 1.26E+05 2.08E+05 2.67E+05 9.82E+04 2.11E+05 2.38E+05 1.37E+05 1.69E+05 Glutathione 1.99E+05 2.35E+05 1.15E+05 1.32E+05 1.51E+05 3.82E+05 6.03E+05 1.42E+05 2.10E+05 3.66E+05 4.41E+05 4.56E+05 5.32E+05 3.88E+05 7.74E+05 9.09E+05 4.50E+05 5.93E+05 Ascorbate 1.90E+05 2.84E+05 2.17E+05 2.34E+05 2.69E+05 1.87E+05 2.59E+05 2.83E+05 1.79E+05 1.74E+05 1.31E+05 1.99E+05 2.28E+05 1.48E+05 2.20E+05 2.50E+05 1.50E+05 1.65E+05 Dehydroascorbate 6.93E+04 7.20E+04 6.98E+04 6.90E+04 5.77E+04 7.42E+04 1.26E+05 8.01E+04 7.54E+04 6.68E+04 9.45E+04 8.84E+04 7.34E+04 9.00E+04 1.13E+05 1.46E+05 1.02E+05 1.30E+05 L-gamma-Glutamyl-L-hypoglycin 3.61E+04 7.03E+04 2.00E+04 3.52E+04 4.62E+04 2.67E+04 6.21E+04 2.77E+04 5.74E+04 3.18E+04 3.84E+04 4.99E+04 4.68E+04 3.40E+04 7.73E+04 4.93E+04 4.03E+04 2.99E+04 1-4-beta-D-Xylan 3.82E+05 4.83E+05 3.81E+05 4.59E+05 3.90E+05 3.57E+05 5.59E+05 5.21E+05 4.17E+05 3.70E+05 3.65E+05 3.84E+05 4.19E+05 3.87E+05 3.77E+05 5.49E+05 2.96E+05 3.71E+05 UDP-N-acetyl-D-glucosamine 5.03E+04 6.17E+04 6.43E+04 6.79E+04 4.75E+04 7.72E+04 8.87E+04 6.20E+04 7.94E+04 9.09E+04 8.69E+04 6.88E+04 7.64E+04 8.47E+04 7.42E+04 9.36E+04 9.16E+04 6.78E+04 Glycolate 5.39E+06 1.64E+06 4.14E+06 6.30E+06 1.39E+06 2.86E+06 1.99E+06 1.79E+06 5.43E+06 5.91E+06 5.06E+06 2.10E+06 2.18E+06 3.84E+06 1.89E+06 2.27E+06 5.89E+06 2.97E+06 Phosphocreatine 1.19E+05 8.86E+04 1.10E+05 1.09E+05 7.17E+04 1.24E+05 1.40E+05 6.05E+04 5.71E+04 2.10E+05 2.53E+05 2.78E+05 2.66E+05 2.77E+05 2.63E+05 3.70E+05 2.02E+05 2.38E+05 Taurine 7.50E+05 6.20E+05 4.38E+05 5.49E+05 5.17E+05 1.24E+06 9.78E+05 4.28E+05 5.89E+05 1.12E+06 1.34E+06 1.38E+06 1.52E+06 1.47E+06 1.69E+06 1.41E+06 8.96E+05 1.14E+06 L-Cysteate 2.42E+05 9.51E+04 1.25E+05 2.73E+05 9.40E+04 1.69E+05 9.26E+04 1.01E+05 2.02E+05 2.51E+05 1.78E+05 1.25E+05 1.05E+05 1.78E+05 1.22E+05 1.11E+05 2.35E+05 1.40E+05 Indole-3-acetaldehyde 1.67E+04 2.43E+04 1.86E+04 1.70E+04 2.11E+04 2.99E+04 5.73E+04 8.18E+04 6.40E+04 1.30E+04 3.11E+04 2.09E+04 2.47E+04 1.14E+04 2.65E+04 7.72E+04 6.74E+04 6.35E+04 Anthranilate 7.58E+04 7.05E+04 5.27E+04 5.06E+04 4.97E+04 6.88E+04 7.49E+04 7.62E+04 6.41E+04 5.97E+04 6.57E+04 1.02E+05 7.54E+04 7.74E+04 8.70E+04 7.52E+04 6.52E+04 1.30E+05 Glycerol 3-phosphate 4.76E+04 8.30E+04 8.84E+04 6.27E+04 5.56E+04 1.01E+05 8.48E+04 1.04E+05 1.49E+05 7.73E+04 5.17E+04 7.29E+04 6.71E+04 6.11E+04 7.80E+04 9.24E+04 8.14E+04 9.38E+04 Ethanolamine phosphate 9.99E+05 9.74E+05 8.90E+05 6.98E+05 7.28E+05 1.63E+06 1.17E+06 6.49E+05 1.01E+06 1.21E+06 1.21E+06 1.32E+06 1.23E+06 1.46E+06 1.48E+06 1.56E+06 1.28E+06 1.44E+06 Sphinganine 1-phosphate 3.46E+05 3.81E+05 3.81E+05 3.97E+05 4.24E+05 4.36E+05 4.97E+05 3.60E+05 3.81E+05 3.61E+05 5.42E+05 3.36E+05 3.53E+05 4.52E+05 4.36E+05 3.96E+05 3.58E+05 3.12E+05 Acetyl phosphate 7.91E+04 8.47E+04 8.84E+04 6.79E+04 8.76E+04 9.33E+04 9.29E+04 8.56E+04 6.65E+04 7.14E+04 6.50E+04 8.87E+04 7.14E+04 7.33E+04 8.42E+04 7.21E+04 7.73E+04 9.69E+04 2-Methyleneglutarate 9.37E+05 1.10E+06 7.80E+05 1.01E+06 9.10E+05 7.98E+05 1.02E+06 1.06E+06 8.34E+05 8.11E+05 7.16E+05 9.67E+05 9.20E+05 7.84E+05 8.70E+05 1.15E+06 6.49E+05 7.92E+05 6-Pyruvoyltetrahydropterin 2.79E+04 4.85E+04 3.57E+04 1.71E+04 3.93E+04 4.60E+04 2.70E+04 3.48E+04 2.44E+04 1.60E+04 4.91E+04 1.08E+04 5.19E+04 3.73E+04 2.80E+04 2.60E+04 2.34E+04 2.71E+04 D-glucono-1,5-lactone 4.31E+05 7.63E+05 5.66E+05 5.81E+05 5.95E+05 3.98E+05 4.61E+05 8.47E+05 4.06E+05 3.10E+05 3.34E+05 5.06E+05 4.95E+05 3.22E+05 5.62E+05 5.31E+05 3.23E+05 4.20E+05 Glucosinolate 1.30E+06 9.05E+05 1.02E+06 1.27E+06 8.47E+05 1.21E+06 1.11E+06 9.49E+05 1.19E+06 1.09E+06 1.15E+06 1.21E+06 1.12E+06 1.28E+06 1.30E+06 1.21E+06 1.32E+06 1.48E+06 9-Oxononanoic acid 3.52E+05 6.42E+05 4.46E+05 2.98E+05 6.43E+05 3.82E+05 3.86E+05 4.90E+05 3.70E+05 4.05E+05 5.64E+05 2.99E+05 3.81E+05 3.46E+05 6.96E+05 4.58E+05 4.40E+05 4.56E+05 6-Carboxyhexanoate 9.35E+05 8.45E+05 7.84E+05 8.43E+05 1.01E+06 7.39E+05 9.87E+05 7.71E+05 9.89E+05 7.34E+05 7.77E+05 8.72E+05 8.30E+05 8.48E+05 9.40E+05 9.75E+05 7.28E+05 9.11E+05 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z r.t. ATP C00002 505.9878 0.59 UTP C00075 482.9611 0.58 UDP C00015 402.9999 0.91 IDP C00104 427.0070 0.54 Pyridoxal C00250 166.0498 2.03 UDP-glucose C00029 565.0474 0.56 Phosphate C00009 96.9680 0.61 Diphosphate C00013 176.9348 0.61 D-Glucose C00031 179.0551 0.66 D-Glucose 6-phosphate C00092 259.0221 0.57 D-Fructose 1-6-bisphosphate C00354 338.9884 0.53 D-Glyceraldehyde 3-phosphate/Glycerone phosphate C00118 168.9883 1.74 2/3-Phospho-D-glycerate C00631 184.9846 0.57 Phosphoenolpyruvate C00074 166.9740 0.57 Pyruvate C00022 87.0071 0.67 Lactate C01432 89.0228 0.66 D-Ribose C00121 149.0443 0.66 D-Rhamnose C01684 163.0614 3.59 Citrate C00158 191.0187 0.58 2-Oxoglutarate C00026 145.0130 0.57 Succinate C00042 117.0178 0.61 Fumarate C00122 115.0022 0.59 Malate C00149 133.0128 0.57 Itaconate C00490 129.0178 0.59 2-Hydroxyglutarate/Citramalate C02630 147.0286 0.56 Sedoheptulose 1-phosphate C06222 289.0328 0.58 alpha-D-Ribose 1-phosphate C00620 229.0117 0.66 Glutathione C00051 306.0764 0.64 Ascorbate C00072 175.0237 0.66 Dehydroascorbate C05422 173.0081 0.56 L-gamma-Glutamyl-L-hypoglycin C08280 269.1157 3.61 1-4-beta-D-Xylan C02352 131.0336 0.67 UDP-N-acetyl-D-glucosamine C00043 606.0742 0.55 Glycolate C00160 75.0071 0.68 Phosphocreatine C02305 210.0278 0.56 Taurine C00245 124.0059 0.66 L-Cysteate C00506 167.9960 0.73 Indole-3-acetaldehyde C00637 158.0599 1.98 Anthranilate C00108 136.0390 1.80 Glycerol 3-phosphate C00093 171.0056 0.60 Ethanolamine phosphate C00346 140.0105 0.62 Sphinganine 1-phosphate C01120 380.2551 3.61 Acetyl phosphate C00227 138.9788 0.60 2-Methyleneglutarate C02930 143.0336 0.67 6-Pyruvoyltetrahydropterin C03684 236.0778 1.75 D-glucono-1,5-lactone C00198 177.0394 0.66 Glucosinolate C17260 316.9841 0.74 9-Oxononanoic acid C16322 171.1015 2.57 6-Carboxyhexanoate C02656 159.0664 3.51 METABOLITES_END #END