#METABOLOMICS WORKBENCH sdasari_20150625_9038001_mwtab.txt DATATRACK_ID:329 STUDY_ID:ST000242 ANALYSIS_ID:AN000376 PROJECT_ID:PR000195 VERSION 1 CREATED_ON 2016-09-17 #PROJECT PR:PROJECT_TITLE Metabolimic Analysis of Conditioned Macrophage Media PR:PROJECT_TYPE Untargeted LC-MS Metabolomics PR:PROJECT_SUMMARY We are studying the secreted components from pro-inflammatory macrophages, PR:PROJECT_SUMMARY M1. We use a macrophage at the opposite end of the macrophage phenotype PR:PROJECT_SUMMARY as a control, the anti-inflammatory macrophage, coined M2. M1 macrophage PR:PROJECT_SUMMARY media has a <3 kD factor that reduces survival of a cell of interest in primary PR:PROJECT_SUMMARY This factor is not present in M2 macrophage conditioned media <3 kD, as there PR:PROJECT_SUMMARY no effect on primary cell numbers. The goal of this project is to analyze the PR:PROJECT_SUMMARY present in the M1 conditioned <3 kD media and compare to the M2 conditioned < PR:PROJECT_SUMMARY media. PR:INSTITUTE Mayo Clinic PR:DEPARTMENT Physiology and Biomedical Engineering PR:LAST_NAME Farrugia PR:FIRST_NAME Gianrico PR:ADDRESS 200 First Street SW, Rochester, MN 55905 PR:EMAIL Dasari.Surendra@mayo.edu PR:PHONE 507-284-0513 #STUDY ST:STUDY_TITLE Whole unconditioned medium (Defined culture media, M199),Whole M1 medium,Whole ST:STUDY_TITLE medium ST:STUDY_TYPE differential metabolomics ST:STUDY_SUMMARY Media was flash frozen with N2 and stored at -80 C. Samples can be stored at ST:STUDY_SUMMARY C until use. ~1 ml aliquots. The following media has been provided to the ST:STUDY_SUMMARY core in biological triplicates.Complete (Whole) Media:1) Whole unconditioned ST:STUDY_SUMMARY (Defined culture media, M199)2) Whole M1 medium3) Whole M2 medium ST:INSTITUTE Mayo Clinic ST:DEPARTMENT Physiology and Biomedical Engineering ST:LAST_NAME Farrugia ST:FIRST_NAME Gianrico ST:ADDRESS - ST:EMAIL Dasari.Surendra@mayo.edu ST:PHONE - ST:NUM_GROUPS 1 #SUBJECT SU:SUBJECT_TYPE Human macrophages SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 2 ms5047-2-1 Sample Status:Whole M1 medium SUBJECT_SAMPLE_FACTORS 2 ms5047-2-2 Sample Status:Whole M1 medium SUBJECT_SAMPLE_FACTORS 2 ms5047-2-3 Sample Status:Whole M1 medium SUBJECT_SAMPLE_FACTORS 3 ms5047-3-1 Sample Status:Whole M2 medium SUBJECT_SAMPLE_FACTORS 3 ms5047-3-2 Sample Status:Whole M2 medium SUBJECT_SAMPLE_FACTORS 3 ms5047-3-3 Sample Status:Whole M2 medium SUBJECT_SAMPLE_FACTORS 1 ms5047-1-1 Sample Status: Whole unconditioned medium (Defined culture media, M199) SUBJECT_SAMPLE_FACTORS 1 ms5047-1-2 Sample Status: Whole unconditioned medium (Defined culture media, M199) SUBJECT_SAMPLE_FACTORS 1 ms5047-1-3 Sample Status: Whole unconditioned medium (Defined culture media, M199) #COLLECTION CO:COLLECTION_SUMMARY Whole media CO:SAMPLE_TYPE Media #TREATMENT TR:TREATMENT_SUMMARY Media was flash frozen with N2 and stored at -80 C. Samples can be stored TR:TREATMENT_SUMMARY -80 C until use. ~1 ml aliquots. The following media has been provided to TR:TREATMENT_SUMMARY metabolomics core in biological triplicates. Complete (Whole) Media: 1) TR:TREATMENT_SUMMARY unconditioned macrophage medium (Defined culture media, M199) 2) Whole M1 TR:TREATMENT_SUMMARY medium 3) Whole M2 macrophage medium #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Quality-control samples used in the study were prepared from pooled samples SP:SAMPLEPREP_SUMMARY with a selection of standards. All samples (200 μL) were thawed on ice at 4°C SP:SAMPLEPREP_SUMMARY by deproteinization with methanol (1:4 ratio of plasma to methanol) and SP:SAMPLEPREP_SUMMARY for 10 s, followed by incubation at −20°C for 2 h. The samples were then SP:SAMPLEPREP_SUMMARY at 15,871g for 30 min at 4°C. The supernatants were lyophilized (Savant, SP:SAMPLEPREP_SUMMARY NY) and stored at −20°C prior to analysis. The samples were reconstituted in SP:SAMPLEPREP_SUMMARY H2O/acetonitrile and passed through a Microcon YM3 filter (Millipore SP:SAMPLEPREP_SUMMARY The supernatants were transferred to analytical vials, stored in the SP:SAMPLEPREP_SUMMARY at 4°C, and analyzed within 48 h of reconstitution in buffer. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY C18 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME high-strength silica 2.1 150 mm, 1.8 m; Waters CH:CHROMATOGRAPHY_COMMENTS Metabolite separation was achieved using an Acquity UPLC system (Waters, CH:CHROMATOGRAPHY_COMMENTS MA) with both hydrophilic interaction chromatography (HILIC) (ethylene-bridged CH:CHROMATOGRAPHY_COMMENTS 2.1150 mm, 1.7 mm; Waters) and reversed-phase liquid chromatography C18 (RPLC) CH:CHROMATOGRAPHY_COMMENTS silica 2.1150 mm, 1.8 m; Waters). For each column, the run time was 20 min at a CH:CHROMATOGRAPHY_COMMENTS rate of 400 L/min. Reverse-phase chromatography was performed using 99% solvent CH:CHROMATOGRAPHY_COMMENTS (5 mmol/L NH4 acetate, 0.1% formic acid, and 1% acetonitrile) to 100% solvent B CH:CHROMATOGRAPHY_COMMENTS acetonitrile with 0.1% formic acid). The gradient was 0 min, 0% B; 1 min, 0% B; CH:CHROMATOGRAPHY_COMMENTS min, 5% B; 13.0 min, 100% B; 16 min, 100% B; 16.5 min, 0% B; and 20 min, 0% B. CH:CHROMATOGRAPHY_COMMENTS hydrophilic interaction chromatography gradient was as follows: 0 min, 100% B; CH:CHROMATOGRAPHY_COMMENTS min, 100% B; 5 min, 90% B; 13.0 min, 0% B; 16 min, 0% B; 16.5 min, 100% B; and CH:CHROMATOGRAPHY_COMMENTS min, 100% B. The injection volume of each sample was 5 L and column was CH:CHROMATOGRAPHY_COMMENTS at 50C. #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6220 TOF MS:INSTRUMENT_TYPE TOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS C18-NESI (-ve ESI) A 6220 ToF-MS (Agilent Technologies) was operated in both MS:MS_COMMENTS and negative electrospray ionization (ESI) modes using a scan range of MS:MS_COMMENTS m/z. The mass accuracy and mass resolution were 5 parts per million (ppm) and MS:MS_COMMENTS ppm, respectively. The instrument settings were as follows: nebulizer gas MS:MS_COMMENTS 325°C, capillary voltage 3.5 kV, capillary temperature 300°C, fragmentor MS:MS_COMMENTS 150 V, skimmer voltage 58 V, octapole voltage 250 V, cycle time 0.5 s, and run MS:MS_COMMENTS 15.0 min. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples ms5047-2-1 ms5047-2-2 ms5047-2-3 ms5047-3-1 ms5047-3-2 ms5047-3-3 ms5047-1-1 ms5047-1-2 ms5047-1-3 Factors Sample Status:Whole M1 medium Sample Status:Whole M1 medium Sample Status:Whole M1 medium Sample Status:Whole M2 medium Sample Status:Whole M2 medium Sample Status:Whole M2 medium Sample Status: Whole unconditioned medium (Defined culture media, M199) Sample Status: Whole unconditioned medium (Defined culture media, M199) Sample Status: Whole unconditioned medium (Defined culture media, M199) C17 H19 N16 O5 11.9964 12.0697 3.7868 12.2598 12.0578 0.0000 0.0000 0.0000 0.0000 C20 H39 N10 O13 14.6761 14.9019 13.6728 14.3355 14.4106 0.0000 0.0000 0.0000 0.0000 C22 H34 N13 O12 S 14.3734 14.4815 12.9978 14.6772 14.4098 0.0000 0.0000 0.0000 0.0000 Camoensine 12.5956 12.6332 11.8163 12.7274 12.5752 0.0000 0.0000 0.0000 0.0000 Cystathionine ketimine 0.0000 0.0000 12.5985 0.0000 11.6463 12.1156 0.0000 0.0000 11.4144 D-(+)-Glyceraldehyde 16.6652 17.0194 16.6422 15.9442 15.9403 15.3867 0.0000 0.0000 0.0000 F-Honaucin A 15.1714 15.3252 15.2866 14.9313 15.0608 14.8516 0.0000 0.0000 0.0000 Glycolaldehyde 15.2608 15.3133 12.6404 15.6615 15.7268 15.2701 15.4650 16.1930 15.6593 Kaempferol 3-sophoroside-7-(2^^-ferulylglucoside) 12.5933 12.6677 12.2817 12.9170 12.6531 0.0000 0.0000 0.0000 0.0000 N-Succinyl-L-diaminopimelic acid 14.1237 14.2983 13.7604 14.3256 14.1788 0.0000 0.0000 0.0000 0.0000 Oxonitine 13.0685 13.1510 12.3430 12.7679 12.9568 0.0000 0.0000 0.0000 0.0000 Penicillin G 15.3646 15.5969 14.8153 15.5630 15.3376 0.0000 0.0000 0.0000 0.0000 Penicilloic G acid 14.9729 15.1315 13.7618 15.3851 15.0568 0.0000 0.0000 0.0000 0.0000 Phenylacetylglycine 12.3805 12.4555 3.7998 12.7646 12.5473 0.0000 0.0000 0.0000 0.0000 Tyrphostin B44 (-) 15.1918 15.3178 14.5218 15.4848 15.2573 0.0000 0.0000 0.0000 0.0000 967.008@1.264 13.0389185 14.861571 0 16.35702 16.449455 0 14.15181 0 12.218564 967.008@1.8880004 0 15.45208 15.102468 15.285041 0 0 16.044907 13.720458 16.276033 207.9797@0.8050001 15.220416 14.100744 16.84976 14.659438 16.17143 16.740782 14.417061 0 16.246164 97.9673@0.82699984 0 16.342594 14.596072 16.412767 0 14.342005 14.104517 0 14.540371 956.9776@0.9060001 14.204647 15.156635 14.303852 13.354112 12.979604 15.325551 15.957329 13.757181 14.478706 181.074@1.5839998 14.732221 14.593858 13.847156 14.595608 14.930138 14.033594 14.7831 15.61186 15.407999 131.0943@2.1720006 15.030366 15.118333 14.139551 15.281965 15.351526 15.081733 14.878434 15.597412 15.543031 165.0789@3.4020002 13.958643 13.9324 13.060696 14.395333 14.572878 14.184797 13.839203 14.622452 14.585901 90.0322@0.9330001 14.089533 14.13571 13.314442 14.397273 14.418643 14.263856 14.251853 14.613329 14.44262 216.0407@0.92200035 13.871136 14.104926 13.323337 14.212193 14.104354 13.96145 14.027647 14.646109 14.105008 956.9774@2.0099993 13.683542 0 0 0 0 0 0 13.514098 0 131.0943@1.8850001 0 0 0 14.146647 0 0 0 14.413959 14.367278 135.054@1.3659999 13.402745 0 0 0 13.823069 0 13.980585 14.460904 14.430059 958.9757@0.9060001 11.845881 13.213863 12.544965 12.610103 0 12.353147 14.200362 11.963618 0 147.054@0.9249998 13.574001 13.655866 12.902752 13.596772 13.557464 13.374633 13.301639 13.693705 13.429668 956.9769@1.0160003 0 13.43593 14.217049 0 12.916252 0 0 13.377482 0 204.0894@3.9479985 12.93719 13.080152 12.159555 13.251926 13.352181 13.131536 12.664669 13.380597 13.300066 159.0186@0.95000005 12.222191 0 13.213257 12.241983 12.847057 13.050869 0 0 12.744413 4-Fluoromuconolactone 12.332876 0 13.369869 12.249113 12.828532 13.074308 0 0 12.836247 114.0318@0.9379999 12.645433 0 12.646784 12.691525 12.955649 12.979424 0 0 13.053756 294.1824@9.494001 12.562719 12.521846 12.544482 12.5610485 12.479023 12.545929 12.515206 12.545929 12.520619 102.0319@0.93499994 12.18797 12.227615 11.515699 12.416269 12.46786 12.308623 12.293183 12.611947 12.572463 99.9644@0.8249999 11.734709 12.104271 0 11.925183 11.779309 11.715961 0 11.837627 11.383164 115.9958@0.817 11.653741 11.579315 0 0 0 11.929628 0 0 11.735133 308.119@5.9570003 16.630152 16.606709 15.326042 16.990812 16.675129 0 0 0 0 308.119@5.3300014 15.678628 15.661945 14.769683 15.801304 15.621279 0 0 0 0 956.9776@1.33 11.912889 0 0 11.326429 0 0 0 0 0 1011.0322@1.3729998 13.910268 14.092674 13.903505 13.506556 13.430714 13.038748 0 0 0 Kaempferol 3-sophoroside-7-(2-ferulylglucoside) 12.647908 12.622051 12.2662 12.733227 12.659327 0 0 0 0 187.0525@1.3729998 12.923698 13.0235815 12.912328 12.760928 12.731956 12.498101 0 0 0 230.1419@5.4079995 12.40939 12.363862 11.721099 12.546171 12.48079 0 0 0 0 958.975@1.3279996 0 0 0 0 0 0 0 0 0 1041.0424@0.76700014 0 12.379378 0 11.729197 11.610102 11.4476 0 0 0 1034.9916@0.7589999 0 0 0 0 0 0 0 0 0 638.2193@5.958 0 0 0 11.466586 0 0 0 0 0 1009.0153@1.0550001 0 0 0 0 0 0 0 0 0 428.1612@5.9550004 0 0 0 0 0 0 0 0 0 706.133@6.002999 0 0 0 0 0 0 0 0 0 179.9848@0.80100006 0 0 14.78484 0 14.145057 0 0 0 14.033681 131.0231@0.95100015 0 0 11.5919895 0 0 0 0 0 0 404.3126@13.796998 11.791163 0 0 0 0 0 11.9783535 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name moverz_quant ri ri_type pubchem_id inchi_key kegg_id other_id other_id_type C17 H19 N16 O5 6.5199995 Mayo_RI MAYO_ID C20 H39 N10 O13 0.7719999 Mayo_RI MAYO_ID C22 H34 N13 O12 S 5.331 Mayo_RI MAYO_ID Camoensine 5.333001 Mayo_RI C10758 MAYO_ID Cystathionine ketimine 0.95299983 Mayo_RI MAYO_ID D-(+)-Glyceraldehyde 1.3720001 Mayo_RI C00577 MAYO_ID F-Honaucin A 1.3720001 Mayo_RI MAYO_ID Glycolaldehyde 0.9330001 Mayo_RI C00266 MAYO_ID Kaempferol 3-sophoroside-7-(2''-ferulylglucoside) 5.9550014 Mayo_RI MAYO_ID N-Succinyl-L-diaminopimelic acid 4.612 Mayo_RI C04421 MAYO_ID Oxonitine 0.77799994 Mayo_RI MAYO_ID Penicillin G 4.612 Mayo_RI C05551 MAYO_ID Penicilloic G acid 5.4079995 Mayo_RI C16672 MAYO_ID Phenylacetylglycine 5.1129994 Mayo_RI C05598 MAYO_ID Tyrphostin B44 (-) 5.4069996 Mayo_RI MAYO_ID 967.008@1.264 967.008 1.264 967.008@1.8880004 967.008 1.8880004 207.9797@0.8050001 207.9797 0.8050001 97.9673@0.82699984 97.9673 0.82699984 956.9776@0.9060001 956.9776 0.9060001 181.074@1.5839998 181.074 1.5839998 131.0943@2.1720006 131.0943 2.1720006 165.0789@3.4020002 165.0789 3.4020002 90.0322@0.9330001 90.0322 0.9330001 216.0407@0.92200035 216.0407 0.92200035 956.9774@2.0099993 956.9774 2.0099993 131.0943@1.8850001 131.0943 1.8850001 135.054@1.3659999 135.054 1.3659999 958.9757@0.9060001 958.9757 0.9060001 147.054@0.9249998 147.054 0.9249998 956.9769@1.0160003 956.9769 1.0160003 204.0894@3.9479985 204.0894 3.9479985 159.0186@0.95000005 159.0186 0.95000005 4-Fluoromuconolactone 160.0154 0.95100015 C16476 114.0318@0.9379999 114.0318 0.9379999 294.1824@9.494001 294.1824 9.494001 102.0319@0.93499994 102.0319 0.93499994 99.9644@0.8249999 99.9644 0.8249999 115.9958@0.817 115.9958 0.817 308.119@5.9570003 308.119 5.9570003 308.119@5.3300014 308.119 5.3300014 956.9776@1.33 956.9776 1.33 1011.0322@1.3729998 1011.0322 1.3729998 Kaempferol 3-sophoroside-7-(2-ferulylglucoside) 978.2745 5.9550014 187.0525@1.3729998 187.0525 1.3729998 230.1419@5.4079995 230.1419 5.4079995 958.975@1.3279996 958.975 1.3279996 1041.0424@0.76700014 1041.0424 0.76700014 1034.9916@0.7589999 1034.9916 0.7589999 638.2193@5.958 638.2193 5.958 1009.0153@1.0550001 1009.0153 1.0550001 428.1612@5.9550004 428.1612 5.9550004 706.133@6.002999 706.133 6.002999 179.9848@0.80100006 179.9848 0.80100006 131.0231@0.95100015 131.0231 0.95100015 404.3126@13.796998 404.3126 13.796998 METABOLITES_END #END