#METABOLOMICS WORKBENCH swalmsle89_20170421_130705 DATATRACK_ID:894 STUDY_ID:ST000626 ANALYSIS_ID:AN000958 PROJECT_ID:PR000456 VERSION 1 CREATED_ON June 22, 2017, 3:55 pm #PROJECT PR:PROJECT_TITLE A strategy for producing a searchable bronchoalveolar lavage fluid compound PR:PROJECT_TITLE database PR:PROJECT_TYPE MS PR:PROJECT_SUMMARY Comprehensive metabolomic databases for Lipid(+/-) and Aqueous(+) fractions of PR:PROJECT_SUMMARY BALF assembled from a mouse and human COPD experiment. PR:INSTITUTE University of Colorado, Denver PR:DEPARTMENT Pharmaceutical Sciences, Anschutz Medical Campus PR:LABORATORY Metabolomics and Mass Spectrometry PR:LAST_NAME Residorph PR:FIRST_NAME Nichole PR:ADDRESS 12850 East Montview Blvd, Denver, CO 80204 PR:EMAIL nichole.reisdorph@ucdenver.edu PR:PHONE - PR:FUNDING_SOURCE P20-HL113445 Metabolic Profiles of COPD phenotypes #STUDY ST:STUDY_TITLE Non targeted metabolomic analysis of mouse and human bronchoalveolar lavage ST:STUDY_TITLE fluid, Lipid(-) experiment ST:STUDY_TYPE Comprehensive profile ST:STUDY_SUMMARY Mouse BALF was collected from C57BL/6 mice (n = 10). Five mice were exposed to ST:STUDY_SUMMARY ambient air for one day (air control) and five mice were exposed to CS for nine ST:STUDY_SUMMARY months (smoking). Human BALF was collected from subjects from the COPDGene ST:STUDY_SUMMARY cohort (n = 5). COPD diagnosis was based on the ratio of forced expiratory ST:STUDY_SUMMARY volume in 1 second to forced vital capacity (FEV1/FVC). Subjects were 45-70 ST:STUDY_SUMMARY years old, BMI 27-45, weight 76-125kg, and were categorized as follows: 1 male ST:STUDY_SUMMARY former smoker without COPD (FEV1/FVC = 0.82), 2 male current smokers without ST:STUDY_SUMMARY COPD (FEV1/FVC = 0.91 and 0.79), 1 female current smoker with moderate COPD ST:STUDY_SUMMARY (FEV1/FVC = 0.51), and 1 male current smoker with moderate COPD (FEV1/FVC = ST:STUDY_SUMMARY 0.64) ST:INSTITUTE University of Colorado Anschutz Medical Campus ST:DEPARTMENT Pharmaceutical Sciences ST:LABORATORY Metabolomics and Mass Spectrometry ST:LAST_NAME Residorph ST:FIRST_NAME Nichole ST:ADDRESS 12850 East Montview Blvd, Denver, CO 80204 ST:EMAIL nichole.reisdorph@ucdenver.edu ST:PHONE - ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 36 #SUBJECT SU:SUBJECT_TYPE Human;Animal SU:SUBJECT_SPECIES Homo sapiens;Mus musculus SU:TAXONOMY_ID 9606 | 10093 SU:GENOTYPE_STRAIN Animal: C57BL/6 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 10031 14442G_BALF_neg_A Species:Human | SmokingStatus:Smoker | Phenotype:Healthy Gender=M; Replicate=A; Spike=- SUBJECT_SAMPLE_FACTORS 10031 14442G_BALF_neg_B Species:Human | SmokingStatus:Smoker | Phenotype:Healthy Gender=M; Replicate=B; Spike=- SUBJECT_SAMPLE_FACTORS 10031 14442G_BALF_neg_C Species:Human | SmokingStatus:Smoker | Phenotype:Healthy Gender=M; Replicate=C; Spike=- SUBJECT_SAMPLE_FACTORS 11833 17800V_BALF_neg_A Species:Human | SmokingStatus:Smoker | Phenotype:COPD Gender=M; Replicate=A; Spike=- SUBJECT_SAMPLE_FACTORS 11833 17800V_BALF_neg_B Species:Human | SmokingStatus:Smoker | Phenotype:COPD Gender=M; Replicate=B; Spike=- SUBJECT_SAMPLE_FACTORS 11833 17800V_BALF_neg_C Species:Human | SmokingStatus:Smoker | Phenotype:COPD Gender=M; Replicate=C; Spike=- SUBJECT_SAMPLE_FACTORS MET106 BALF_MET106_4x_A Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=A; Spike=4x SUBJECT_SAMPLE_FACTORS MET106 BALF_MET106_4x_B Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=B; Spike=4x SUBJECT_SAMPLE_FACTORS MET106 BALF_MET106_4x_C Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=C; Spike=4x SUBJECT_SAMPLE_FACTORS MET107 BALF_MET107_1x_A Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=A; Spike=1x SUBJECT_SAMPLE_FACTORS MET107 BALF_MET107_1x_B Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=B; Spike=1x SUBJECT_SAMPLE_FACTORS MET107 BALF_MET107_1x_C Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=C; Spike=1x SUBJECT_SAMPLE_FACTORS MET108 BALF_MET108_2x_A Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=A; Spike=2x SUBJECT_SAMPLE_FACTORS MET108 BALF_MET108_2x_B Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=B; Spike=2x SUBJECT_SAMPLE_FACTORS MET108 BALF_MET108_2x_C Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=C; Spike=2x SUBJECT_SAMPLE_FACTORS MET126 BALF_MET126_4x_A Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=A; Spike=4x SUBJECT_SAMPLE_FACTORS MET126 BALF_MET126_4x_B Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=B; Spike=4x SUBJECT_SAMPLE_FACTORS MET126 BALF_MET126_4x_C Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=C; Spike=4x SUBJECT_SAMPLE_FACTORS MET128 BALF_MET128_1x_A Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=A; Spike=1x SUBJECT_SAMPLE_FACTORS MET128 BALF_MET128_1x_B Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=B; Spike=1x SUBJECT_SAMPLE_FACTORS MET128 BALF_MET128_1x_C Species:Mouse | SmokingStatus:Smoker | Phenotype:Emphysema Gender=F; Replicate=C; Spike=1x SUBJECT_SAMPLE_FACTORS MET133 BALF_MET133_1x_A Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=A; Spike=1x SUBJECT_SAMPLE_FACTORS MET133 BALF_MET133_1x_B Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=B; Spike=1x SUBJECT_SAMPLE_FACTORS MET133 BALF_MET133_1x_C Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=C; Spike=1x SUBJECT_SAMPLE_FACTORS MET141 BALF_MET141_2x_A Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=A; Spike=2x SUBJECT_SAMPLE_FACTORS MET141 BALF_MET141_2x_B Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=B; Spike=2x SUBJECT_SAMPLE_FACTORS MET141 BALF_MET141_2x_C Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=C; Spike=2x SUBJECT_SAMPLE_FACTORS MET142 BALF_MET142_4x_A Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=A; Spike=4x SUBJECT_SAMPLE_FACTORS MET142 BALF_MET142_4x_B Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=B; Spike=4x SUBJECT_SAMPLE_FACTORS MET142 BALF_MET142_4x_C Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=C; Spike=4x SUBJECT_SAMPLE_FACTORS MET143 BALF_MET143_1x_A Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=A; Spike=1x SUBJECT_SAMPLE_FACTORS MET143 BALF_MET143_1x_B Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=B; Spike=1x SUBJECT_SAMPLE_FACTORS MET143 BALF_MET143_1x_C Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=C; Spike=1x SUBJECT_SAMPLE_FACTORS MET144 BALF_MET144_2x_A Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=A; Spike=2x SUBJECT_SAMPLE_FACTORS MET144 BALF_MET144_2x_B Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=B; Spike=2x SUBJECT_SAMPLE_FACTORS MET144 BALF_MET144_2x_C Species:Mouse | SmokingStatus:Non-Smoker | Phenotype:Healthy Gender=F; Replicate=C; Spike=2x #COLLECTION CO:COLLECTION_SUMMARY Mouse BALF collection was performed using a total of 1.0 mL PBS divided into CO:COLLECTION_SUMMARY three washes and stored at -80°C until analysis. Human BALF was collected in CO:COLLECTION_SUMMARY the right middle lobe and lingual by instilling two aliquots of 40 mL and one CO:COLLECTION_SUMMARY aliquot of 50 mL of sterile saline per lobe (i.e., 130 mL per lobe, total CO:COLLECTION_SUMMARY volume = 260 mL per subject), which is withdrawn by gentle manual suction CO:COLLECTION_SUMMARY and immediately placed on ice. Aliquots for metabolomics analysis were frozen at CO:COLLECTION_SUMMARY -80 °C and stored until sample preparation and MS analysis. CO:SAMPLE_TYPE BALF CO:COLLECTION_METHOD Broncheoalveolar lavage #TREATMENT TR:TREATMENT_SUMMARY None #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 100uL of BALF were prepared using methanol assisted protein precipitation SP:SAMPLEPREP_SUMMARY followed by methyl-tert butyl ether (MTBE) liquid-liquid extraction. Both liquid SP:SAMPLEPREP_SUMMARY and aqueous fractions were preserved for analysis. The lipid fraction was SP:SAMPLEPREP_SUMMARY reconstituted in 200µL of methanol. The lipid fraction was dried down in a SP:SAMPLEPREP_SUMMARY SpeedVac at 45°C for reconstitution in 50 µL of 95:5 water:acetonitrile. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The BALF samples were separated using C18 reverse phase chromatography. The CH:CHROMATOGRAPHY_SUMMARY separations used an Agilent Zorbax Rapid Resolution HD (RRHD) SB-C18, 1.8 micron CH:CHROMATOGRAPHY_SUMMARY (2.1 x 100 mm) analytical column with an Agilent Zorbax SB-C18, 1.8 micron (2.1 CH:CHROMATOGRAPHY_SUMMARY x 5 mm) guard column and used an Agilent 1290 series high performance liquid CH:CHROMATOGRAPHY_SUMMARY chromatography (HPLC) pump. Injection volumes were 4µL for mouse BALF and 15µL CH:CHROMATOGRAPHY_SUMMARY for human BALF. Flow rate was 0.7 mL/min using the following mobile phases: CH:CHROMATOGRAPHY_SUMMARY mobile phase A was water with 0.1% formic acid, and mobile phase B was 60:36:4 CH:CHROMATOGRAPHY_SUMMARY isopropyl alcohol:acetonitrile:water with 0.1% formic acid. Gradients were 0-0.5 CH:CHROMATOGRAPHY_SUMMARY minutes 30-70% B, 0.5-7.42 minutes 70-100% B, 7.42-9.9 minutes 100% B, 9.9-10.0 CH:CHROMATOGRAPHY_SUMMARY minutes 100-30% B, 10-14.6 minutes 30% B for the positive mode injections. For CH:CHROMATOGRAPHY_SUMMARY negative mode the following eluant profile was used: 0-1 minutes 30-70% B, CH:CHROMATOGRAPHY_SUMMARY 1-7.92 minutes 70-100% B, 7.92-10.4 minutes 100% B, 10.4-10.5 minutes 100-30% B, CH:CHROMATOGRAPHY_SUMMARY 10.5-15.1 minutes 30% B. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 CH:COLUMN_NAME Agilent Zorbax RRHD SB-C18 (100 x 2.1mm, 1.8um) CH:COLUMN_NAME analytical column with an Agilent Zorbax SB-C18, 1.8 micron (2.1 x 5 mm) guard CH:COLUMN_NAME column CH:FLOW_RATE 0.7ml/min #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6510 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:CAPILLARY_VOLTAGE 4000V MS:FRAGMENT_VOLTAGE 120 MS:DESOLVATION_GAS_FLOW 12mL/min MS:DESOLVATION_TEMPERATURE 300C MS:NEBULIZER 30psi MS:SCAN_RANGE_MOVERZ 60-1600 MS:SCANNING 2.02 spectra/second MS:SKIMMER_VOLTAGE 60V MS:MS_RESULTS_FILE ST000626_AN000958_Results.txt UNITS:peak intensity #END