#METABOLOMICS WORKBENCH chichen@umn.edu_20161002_221547_mwtab.txt DATATRACK_ID:750 STUDY_ID:ST000482 ANALYSIS_ID:AN000748 PROJECT_ID:PR000366 VERSION 1 CREATED_ON October 2, 2016, 11:25 pm #PROJECT PR:PROJECT_TITLE Reference plasma sample in CHEAR untargeted resource PR:PROJECT_SUMMARY To determine the chemical composition of reference plasma sample for untargeted PR:PROJECT_SUMMARY 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 Free amino acids in CHEAR reference plasma ST:STUDY_TYPE Quantitative analysis ST:STUDY_SUMMARY To determine the chemical composition of reference plasma sample for untargeted ST:STUDY_SUMMARY 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 1 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 - plasmaRef Sample type:Unknown #COLLECTION CO:COLLECTION_SUMMARY Human Plasma collected and processed by BioreclamationIVT. The plasma pool was CO:COLLECTION_SUMMARY created by combining recovered plasma obtained from 50 males and 50 females with CO:COLLECTION_SUMMARY an average age of 38.4 (range 19-72) and equal distribution of races. CO:SAMPLE_TYPE Plasma #TREATMENT TR:TREATMENT_SUMMARY The plasma pool was created by combining recovered plasma obtained from 50 males TR:TREATMENT_SUMMARY and 50 females with an average age of 38.4 (range 19-72) and equal distribution TR:TREATMENT_SUMMARY of races. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Human reference plasma samples is derivatized with dansyl chloride (DC) prior to SP:SAMPLEPREP_SUMMARY the LC-MS analysis. Briefly, 5 uL of sample or standard was mixed with 5 uL of SP:SAMPLEPREP_SUMMARY of 100 µM indole-D5-L-tryptophan internal standard), 50 uL of 10 mM sodium SP:SAMPLEPREP_SUMMARY carbonate, and 100 uL of DC solution (3 mg/mL in acetone). The mixture was SP:SAMPLEPREP_SUMMARY incubated at 25°C for 15 min and centrifuged at 18,000 × g for 10 min, the SP:SAMPLEPREP_SUMMARY supernatant was transferred into a HPLC vial for 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 plasmaRef Factors Sample type:Unknown Alanine 425.6 Arginine 65.28 Asparagine 18.74 Aspartic acid 8.26 Citruline 27.16 Glutamic Acid 194.06 Glutamine 202.04 Glycine 259.82 Histidine 62.62 Isoleucine 120.64 Leucine 120.64 Lysine 139.22 Methionine 11.84 Ornithine 40 Phenylalanine 88.08 Proline 229.08 Serine 114.76 Taurine 36.78 Threonine 162.48 Tyrosine 43.18 Tryptophan 44.62 Valine 247.22 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID Alanine 0.72 323.1066 5950 Arginine 2.63 408.1706 6322 Asparagine 2.64 366.1124 6267 Aspartic acid 3.03 367.0964 5960 Citruline 2.63 409.1546 9750 Glutamic Acid 2.32 381.112 33032 Glutamine 2.62 380.128 5961 Glycine 0.72 309.0909 750 Histidine 2.3 389.1284 6274 Isoleucine 2.31 365.1535 6306 Leucine 0.71 365.1535 6106 Lysine 2.3 613.2149 5962 Methionine 2.39 399.1048 6137 Ornithine 2.72 599.1998 6262 Phenylalanine 0.71 399.1379 6140 Proline 2.31 349.1222 145742 Serine 0.74 339.1015 5951 Taurine 0.53 359.0735 1123 Threonine 0.7 353.1171 6288 Tyrosine 1.8 648.1838 6057 Tryptophan 0.74 438.1488 6305 Valine 2.64 351.1379 6287 METABOLITES_END #END