#METABOLOMICS WORKBENCH chichen@umn.edu_20170219_143450_mwtab.txt DATATRACK_ID:849 STUDY_ID:ST000557 ANALYSIS_ID:AN000855 PROJECT_ID:PR000408 VERSION 1 CREATED_ON February 21, 2017, 9:52 am #PROJECT PR:PROJECT_TITLE Reference urine sample in CHEAR untargeted resource PR:PROJECT_SUMMARY To determine the chemical composition of reference urine and plasma sample for PR:PROJECT_SUMMARY untargeted metabolomic analysis in CHEAR untargeted resources. PR:INSTITUTE University of Minesota PR:DEPARTMENT Department of Food Science and Nutrition PR:LABORATORY Nutritional Metabolomics PR:LAST_NAME Chen PR:FIRST_NAME Chi PR:ADDRESS 1334 Eckles Ave W, St Paul, MN 55108 PR:EMAIL chichen@umn.edu PR:PHONE 612-624-7704 PR:FUNDING_SOURCE NIEHS #STUDY ST:STUDY_TITLE Metabolites in CHEAR reference urine and plasma ST:STUDY_TYPE Quantitative analysis ST:STUDY_SUMMARY To determine the chemical composition of reference urine and plasma samples for ST:STUDY_SUMMARY untargeted metabolomic analysis in CHEAR untargeted resources. ST:INSTITUTE University of Minesota ST:DEPARTMENT Department of Food Science and Nutrition ST:LABORATORY Nutritional Metabolomics ST:LAST_NAME Chen ST:FIRST_NAME Chi ST:ADDRESS 1334 Eckles Ave W, St Paul, MN 55108 ST:EMAIL chichen@umn.edu ST:PHONE 612-624-7704 ST:NUM_GROUPS 1 ST:TOTAL_SUBJECTS 2 ST:NUM_MALES pooled ST:NUM_FEMALES pooled #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER pooled #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Ref20160721 Sample type:Urine SUBJECT_SAMPLE_FACTORS - Ref20160726 Sample type:Plasma #COLLECTION CO:COLLECTION_SUMMARY Human urine and Plasma collected and processed by BioreclamationIVT. The plasma CO:COLLECTION_SUMMARY pool was created by combining recovered plasma obtained from 50 males and 50 CO:COLLECTION_SUMMARY females with an average age of 38.4 (range 19-72) and equal distribution of CO:COLLECTION_SUMMARY races. CO:SAMPLE_TYPE urine and Plasma #TREATMENT TR:TREATMENT_SUMMARY The urine and plasma pool was created by combining recovered plasma obtained TR:TREATMENT_SUMMARY from 50 males and 50 females with an average age of 38.4 (range 19-72) and equal TR:TREATMENT_SUMMARY distribution of races. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Urine samples were thawed on ice (thaw/freeze cycle=2) 3 aliquots of 100 uL SP:SAMPLEPREP_SUMMARY urine samples were mixed with 100uL of acetonitrile with 2uM sulfadimethoxine SP:SAMPLEPREP_SUMMARY (internal standard). The mixture was vortexed at 3000rpm for 10s, and SP:SAMPLEPREP_SUMMARY centrifuged at 21000g at 4 oC for 10 min. Supernatant was transferred to HPLC SP:SAMPLEPREP_SUMMARY vials and analyzed by LC/MS. Human reference plasma samples is derivatized with SP:SAMPLEPREP_SUMMARY dansyl chloride (DC) prior to the LC-MS analysis. Briefly, 5 uL of sample or SP:SAMPLEPREP_SUMMARY standard was mixed with 5 uL of of 100 µM indole-D5-L-tryptophan internal SP:SAMPLEPREP_SUMMARY standard), 50 uL of 10 mM sodium carbonate, and 100 uL of DC solution (3 mg/mL SP:SAMPLEPREP_SUMMARY in acetone). The mixture was incubated at 25°C for 15 min and centrifuged at SP:SAMPLEPREP_SUMMARY 18,000 × g for 10 min, the supernatant was transferred into a HPLC vial for SP:SAMPLEPREP_SUMMARY LC-MS analysis. SP:PROCESSING_METHOD derivatization, vortex, centrifugation SP:PROCESSING_STORAGE_CONDITIONS On ice #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY for detecting derivatized amino acids CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity BEH C18 (50 x 2.1mm, 1.7um) CH:FLOW_GRADIENT 0-0.5min-0.5%B, 0.5-4min-gradient to 20%B, 4-8min-gradient to 95%B, CH:FLOW_GRADIENT 8-8.1min-gradient to 100%B, 8.1-9min-100%B, 9-9.1min gradient to 0.5%B, CH:FLOW_GRADIENT 9.1-10min-0.5%B CH:FLOW_RATE 0.5mL/min CH:COLUMN_TEMPERATURE 40 C CH:SOLVENT_A Water with 0.1% formic acid CH:SOLVENT_B Acetonitrile with 0.1% formic acid CH:COLUMN_PRESSURE 15000psi (Max) CH:SAMPLE_INJECTION 5 uL CH:ANALYTICAL_TIME 10min CH:WEAK_WASH_SOLVENT_NAME 50% methanol in water CH:WEAK_WASH_VOLUME 500 uL CH:STRONG_WASH_SOLVENT_NAME 50% isopropanol, 25% acetonitrile and 25% water CH:STRONG_WASH_VOLUME 500 uL CH:SAMPLE_LOOP_SIZE 20 uL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-G2-S MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MASS_ACCURACY <5 ppm #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS uM MS_METABOLITE_DATA_START Samples Ref20160721 Ref20160726 Factors Sample type:Urine Sample type:Plasma L-Alanine 86.4 425.6 L-Asparagine 23.3 18.7 L-Aspartic acid 8.1 8.3 L-Glutamic acid 58.8 194.1 L-Glutamine 4.3 202 Glycine 1198.6 259.8 L-Isoleucine/Leucine 1.1 120.6 L-Methionine 3.0 11.8 L-Phenylalanine 28.4 88.1 L-Proline 4.3 229.1 L-Serine 73.5 114.8 Taurine 991.7 36.8 L-Threonine 69.4 162.5 L-Tyrosine 18.9 43.2 L-Valine 13.1 247.2 L-Arginine 3.7 65.3 L-Lysine 39.8 139.2 L-Histidine 418.8 62.6 L-Tryptophan 33.9 44.6 L-Ornithine 4.6 40 4-Aminobutyric acid 1.7 0 Carnosine 16.1 0.18 Benzoic acid 17.3 0.9 Formic acid 103.0 20.5 Glutaric acid 3.5 0.1 3-Hydroxyburtyric acid 0.0 27.5 o-Hydroxyphenylacetic acid 181.0 0.2 Indoleacetic acid 26.6 0.7 Lactic acid 746.7 1329.1 Pyruvic acid 1906.5 56.3 suberic acid 1.4 0.0 Creatinine 10010 22.2 Phenylacetylglutamine 789.0 0 Hypoxanthine 1.5 1.3 Kynurenic acid 0.9 0.1 Xanthurenic acid 0.5 0 Sebacic acid 1.3 0.1 Threonic acid 23.5 4.6 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID L-Alanine 5.03 323.1066 5950 L-Asparagine 3.71 366.1124 6267 L-Aspartic acid 4.47 367.0964 5960 L-Glutamic acid 4.47 381.112 33032 L-Glutamine 3.99 380.128 5961 Glycine 4.75 309.0909 750 L-Isoleucine/Leucine 5.97 365.1535 6106 L-Methionine 4.26 399.1048 6137 L-Phenylalanine 5.92 399.1379 6140 L-Proline 5.52 349.1222 145742 L-Serine 4.22 339.1015 5951 Taurine 4.05 359.0735 1123 L-Threonine 4.71 353.1171 6288 L-Tyrosine 6.97 648.1838 6057 L-Valine 5.62 351.1379 6287 L-Arginine 3.99 408.1706 6322 L-Lysine 6.43 613.2149 5962 L-Histidine 6.55 389.1284 6274 L-Tryptophan 5.74 438.1488 6305 L-Ornithine 6.32 599.1998 6262 4-Aminobutyric acid 5.05 337.1222 119 Carnosine 6.41 460.1655 439224 Benzoic acid 4.73 264.1137 243 Formic acid 2.32 188.0824 284 Glutaric acid 2.7 274.1192 743 3-Hydroxyburtyric acid 2.7 246.1243 441 o-Hydroxyphenylacetic acid 3.78 294.1243 11970 Indoleacetic acid 5.1 317.1402 802 Lactic acid 2.58 232.1086 612 Pyruvic acid 3.51 230.093 1060 suberic acid 5.09 316.1661 10457 Creatinine 3.2 114.0667 588 Phenylacetylglutamine 2.6 265.1188 92258 Hypoxanthine 2.39 137.0463 790 Kynurenic acid 2.32 190.0504 3845 Xanthurenic acid 2.47 206.0453 5699 Sebacic acid 0.72 201.1127 5192 Threonic acid 2.47 135.0293 151152 METABOLITES_END #END