#METABOLOMICS WORKBENCH hormel101_20161021_131456_mwtab.txt DATATRACK_ID:762 VERSION 1 CREATED_ON November 16, 2016, 2:50 pm #PROJECT PR:PROJECT_TITLE Metabolic Dissection of Aberrant Glutamine Metabolism in Prostate Cancer PR:PROJECT_TYPE MS targeted analysis PR:PROJECT_SUMMARY In this project, we propose to define the specific role(s) of three crucial PR:PROJECT_SUMMARY oncogenic signaling cascades (androgen receptor, Myc and AMPK) in regulating PR:PROJECT_SUMMARY glutamine metabolism in prostate cancer using 13C-labeled glutamine with mass PR:PROJECT_SUMMARY spectrometry-based metabolic flux analyses. PR:INSTITUTE Mayo Clinic PR:LABORATORY Mayo Clinic Metabolomics Resource Core PR:LAST_NAME Frigo PR:FIRST_NAME Daniel PR:ADDRESS Department of Biology and Biochemistry, University of Houston, Houston, Texas PR:ADDRESS 77204-5001 PR:EMAIL frigo@uh.edu PR:PHONE 832-842-8824 #STUDY ST:STUDY_TITLE Timecourse of palmitate isotopomers in LNCaP Cells ST:STUDY_SUMMARY Timecourse of palmitate isotopomers in LNCaP Cell pellets ST:INSTITUTE Mayo Clinic ST:LABORATORY Mayo Clinic Metabolomics Resource Core ST:LAST_NAME Frigo ST:FIRST_NAME Daniel ST:ADDRESS Department of Biology and Biochemistry, University of Houston, Houston, Texas ST:ADDRESS 77204-5001 ST:EMAIL frigo@uh.edu ST:PHONE 832-842-8824 #SUBJECT SU:SUBJECT_TYPE Human 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 DF-1 ms5457-1 Treatment:treated-vehicle (ethanol) | 13C media incubation time:24 hours Amount=3 million cells; 1x PBS=50; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-2 ms5457-2 Treatment:treated-100 pM R1881 | 13C media incubation time:24 hours Amount=3 million cells; 1x PBS=50; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-3 ms5457-3 Treatment:treated-1 nM R1881 | 13C media incubation time:24 hours Amount=3 million cells; 1x PBS=50; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-4 ms5457-4 Treatment:treated-10 nM R1881 | 13C media incubation time:24 hours Amount=3 million cells; 1x PBS=50; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-8 ms5457-5 Treatment:treated-vehicle (ethanol) | 13C media incubation time:72 hours Amount=3 million cells; 1x PBS=50; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-9 ms5457-6 Treatment:treated-100 pM R1881 | 13C media incubation time:72 hours Amount=3 million cells; 1x PBS=50; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-10 ms5457-7 Treatment:treated-1 nM R1881 | 13C media incubation time:72 hours Amount=3 million cells; 1x PBS=50; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-11 ms5457-8 Treatment:treated-10 nM R1881 | 13C media incubation time:72 hours Amount=3 million cells; 1x PBS=50; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-1 ms5457-9 Treatment:treated-vehicle (ethanol) | 13C media incubation time:24 hours Amount=10 mls spent media; 1x PBS=-; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-2 ms5457-10 Treatment:treated-100 pM R1881 | 13C media incubation time:24 hours Amount=10 mls spent media; 1x PBS=-; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-3 ms5457-11 Treatment:treated-1 nM R1881 | 13C media incubation time:24 hours Amount=10 mls spent media; 1x PBS=-; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-4 ms5457-12 Treatment:treated-10 nM R1881 | 13C media incubation time:24 hours Amount=10 mls spent media; 1x PBS=-; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-8 ms5457-13 Treatment:treated-vehicle (ethanol) | 13C media incubation time:72 hours Amount=10 mls spent media; 1x PBS=-; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-9 ms5457-14 Treatment:treated-100 pM R1881 | 13C media incubation time:72 hours Amount=10 mls spent media; 1x PBS=-; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-10 ms5457-15 Treatment:treated-1 nM R1881 | 13C media incubation time:72 hours Amount=10 mls spent media; 1x PBS=-; assay=palmitate isotopomers SUBJECT_SAMPLE_FACTORS DF-11 ms5457-16 Treatment:treated-10 nM R1881 | 13C media incubation time:72 hours Amount=10 mls spent media; 1x PBS=-; assay=palmitate isotopomers #COLLECTION CO:COLLECTION_SUMMARY LNCaP cell model #TREATMENT TR:TREATMENT_SUMMARY LNCaP cells that were serum starved and incubated with 2mM [U-13C]glutamine and TR:TREATMENT_SUMMARY treated with dose response of a synthetic androgen (R1881) for 24 or 72 hours. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Add 50 uL 1x PBS solution to each vial containing cells. Cap, vortex, sonicate 2 SP:SAMPLEPREP_SUMMARY x 20 sec bursts with vortexing inbetween and after Transfer entire contents of SP:SAMPLEPREP_SUMMARY each vial into a labeled 1 dram vial. use 50 uL media for samples 9-16 use 50 uL SP:SAMPLEPREP_SUMMARY plasma for QC Add 1.5 mL CHM solution to vial Add 20 uL 1:20 phenol red SP:SAMPLEPREP_SUMMARY indicator Cap and vortex on multivortexer for 10 minutes Centrifuge @ 4C @ 3000 SP:SAMPLEPREP_SUMMARY rpm for 10 minutes Aspirate and discard top phase hard to see the phase SP:SAMPLEPREP_SUMMARY separation due to the light color (yellow). Dry bottom phase in speed vac SP:SAMPLEPREP_SUMMARY Reconstitute samples and standards with 25 uL buffer A. (do triplicate SP:SAMPLEPREP_SUMMARY injections) #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters Acquity BEH C18 (150 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6460 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS 13C-palmitate % enrichment MS_METABOLITE_DATA_START Samples ms5457-1 ms5457-2 ms5457-3 ms5457-4 ms5457-5 ms5457-6 ms5457-7 ms5457-8 ms5457-9 ms5457-10 ms5457-11 ms5457-12 ms5457-13 ms5457-14 ms5457-15 ms5457-16 Factors Treatment:treated-vehicle (ethanol) | 13C media incubation time:24 hours Treatment:treated-100 pM R1881 | 13C media incubation time:24 hours Treatment:treated-1 nM R1881 | 13C media incubation time:24 hours Treatment:treated-10 nM R1881 | 13C media incubation time:24 hours Treatment:treated-vehicle (ethanol) | 13C media incubation time:72 hours Treatment:treated-100 pM R1881 | 13C media incubation time:72 hours Treatment:treated-1 nM R1881 | 13C media incubation time:72 hours Treatment:treated-10 nM R1881 | 13C media incubation time:72 hours Treatment:treated-vehicle (ethanol) | 13C media incubation time:24 hours Treatment:treated-100 pM R1881 | 13C media incubation time:24 hours Treatment:treated-1 nM R1881 | 13C media incubation time:24 hours Treatment:treated-10 nM R1881 | 13C media incubation time:24 hours Treatment:treated-vehicle (ethanol) | 13C media incubation time:72 hours Treatment:treated-100 pM R1881 | 13C media incubation time:72 hours Treatment:treated-1 nM R1881 | 13C media incubation time:72 hours Treatment:treated-10 nM R1881 | 13C media incubation time:72 hours m0 palmitate isotopomers 88.99 87.46 87.81 83.06 83.13 77.56 78.33 76.18 99.97 99.83 99.82 99.91 99.69 99.67 99.86 99.99 m1 palmitate isotopomers 0.28 0.65 0.67 1.11 1.67 2.29 2.41 2.31 0.00 0.17 0.16 0.00 0.31 0.33 0.13 0.00 m2 palmitate isotopomers 5.91 5.95 6.14 8.03 9.81 13.05 12.92 14.00 0.03 0.00 0.00 0.08 0.00 0.00 0.00 0.00 m3 palmitate isotopomers 0.31 0.41 0.37 0.56 0.71 0.92 0.68 0.82 0.00 0.00 0.01 0.00 0.00 0.01 0.01 0.01 m4 palmitate isotopomers 2.92 3.44 3.19 4.55 3.34 4.42 4.16 4.76 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m5 palmitate isotopomers 0.08 0.13 0.09 0.15 0.12 0.17 0.10 0.18 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m6 palmitate isotopomers 1.06 1.36 1.18 1.78 0.90 1.21 1.05 1.33 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m7 palmitate isotopomers 0.00 0.00 0.00 0.00 0.00 0.01 0.02 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m8 palmitate isotopomers 0.35 0.45 0.44 0.58 0.24 0.30 0.26 0.33 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m9 palmitate isotopomers 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m10 palmitate isotopomers 0.11 0.15 0.13 0.17 0.07 0.08 0.07 0.09 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m11 palmitate isotopomers 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m12 palmitate isotopomers 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m13 palmitate isotopomers 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m14 palmitate isotopomers 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m15 palmitate isotopomers 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m16 palmitate isotopomers 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m0 palmitate isotopomers m1 palmitate isotopomers m2 palmitate isotopomers m3 palmitate isotopomers m4 palmitate isotopomers m5 palmitate isotopomers m6 palmitate isotopomers m7 palmitate isotopomers m8 palmitate isotopomers m9 palmitate isotopomers m10 palmitate isotopomers m11 palmitate isotopomers m12 palmitate isotopomers m13 palmitate isotopomers m14 palmitate isotopomers m15 palmitate isotopomers m16 palmitate isotopomers METABOLITES_END #END