#METABOLOMICS WORKBENCH kenanazam_20160908_161800_mwtab.txt DATATRACK_ID:728 STUDY_ID:ST000459 ANALYSIS_ID:AN000720 PROJECT_ID:PR000354 VERSION 1 CREATED_ON September 8, 2016, 5:11 pm #PROJECT PR:PROJECT_TITLE The effect of Odc knockout on amino acid homeostasis PR:PROJECT_TYPE Amino acid steady-state level analysis PR:PROJECT_SUMMARY YA95 is a lymphoma cell line from a Eμ-Myc;rtTA;Odc fl/fl mouse. The cell line PR:PROJECT_SUMMARY was transduced with a Dox-inducible control vector (E) or a vector containing PR:PROJECT_SUMMARY Cre recombinase (C). Doxycycline treatment promotes excision of Odc gene in YA95 PR:PROJECT_SUMMARY C cells. In this experiment, we are ascertaining the effects of Odc deletion on PR:PROJECT_SUMMARY amino acid homeostasis 48 hours after Dox treatment. PR:INSTITUTE H. Lee Moffitt Cancer Center PR:DEPARTMENT Tumor Biology PR:LABORATORY John Cleveland PR:LAST_NAME Fernandez PR:FIRST_NAME Mario PR:ADDRESS 12902 Magnolia Drive, MRC 4-West PR:EMAIL mario.fernandez@moffitt.org PR:PHONE 813-745-5140 PR:FUNDING_SOURCE 5R01CA154739 #STUDY ST:STUDY_TITLE Effect of Odc knockout on amino acid steady state levels of Eμ-Myc lymphomas ST:STUDY_TYPE Cells are treated with vehicle or Doxycycline - to promote deletion of Odc gene ST:STUDY_SUMMARY Cells were collected 48 hours after treatment with Dox. ST:INSTITUTE University of Florida ST:DEPARTMENT SECIM ST:LABORATORY John Cleveland ST:LAST_NAME Fernandez ST:FIRST_NAME Mario ST:ADDRESS 12902 Magnolia Drive, MRC 4-West ST:EMAIL mario.fernandez@moffitt.org ST:PHONE 813-745-5140 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 12 ST:STUDY_COMMENTS Groups collected in triplicate per time point, either treated or untreated with ST:STUDY_COMMENTS Doxycycline. #SUBJECT SU:SUBJECT_TYPE Cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN Eμ-Myc;rtTA;Odc fl/fl - C57BL/6 SU:CELL_STRAIN_DETAILS YA95 SU:CELL_PRIMARY_IMMORTALIZED Immortalized SU:CELL_PASSAGE_NUMBER 5 SU:CELL_COUNTS 6e6 to 20e6/sample #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS YA95 E YA95_1 Treatment:Vehicle | Genotype:Odc WT Hours=48 SUBJECT_SAMPLE_FACTORS YA95 E YA95_2 Treatment:Vehicle | Genotype:Odc WT Hours=48 SUBJECT_SAMPLE_FACTORS YA95 E YA95_3 Treatment:Vehicle | Genotype:Odc WT Hours=48 SUBJECT_SAMPLE_FACTORS YA95 E YA95_4 Treatment:Doxycycline | Genotype:Odc WT Hours=48 SUBJECT_SAMPLE_FACTORS YA95 E YA95_5 Treatment:Doxycycline | Genotype:Odc WT Hours=48 SUBJECT_SAMPLE_FACTORS YA95 E YA95_6 Treatment:Doxycycline | Genotype:Odc WT Hours=48 SUBJECT_SAMPLE_FACTORS YA95 C YA95_7 Treatment:Vehicle | Genotype:Odc WT Hours=48 SUBJECT_SAMPLE_FACTORS YA95 C YA95_8 Treatment:Vehicle | Genotype:Odc WT Hours=48 SUBJECT_SAMPLE_FACTORS YA95 C YA95_9 Treatment:Vehicle | Genotype:Odc WT Hours=48 SUBJECT_SAMPLE_FACTORS YA95 C YA95_10 Treatment:Doxycycline | Genotype:Odc -/- Hours=48 SUBJECT_SAMPLE_FACTORS YA95 C YA95_11 Treatment:Doxycycline | Genotype:Odc -/- Hours=48 SUBJECT_SAMPLE_FACTORS YA95 C YA95_12 Treatment:Doxycycline | Genotype:Odc -/- Hours=48 #COLLECTION CO:COLLECTION_SUMMARY Cells were counted, cells were collected via centrifugation, washed twice with CO:COLLECTION_SUMMARY cold 1x PBS, PBS removed and flash frozen. Pellets were saved at -80 C. CO:SAMPLE_TYPE Cell CO:COLLECTION_METHOD Pellet CO:COLLECTION_LOCATION Lab CO:COLLECTION_FREQUENCY Once at 48 hours post-treatment CO:VOLUMEORAMOUNT_COLLECTED Depends on sample. Collected as many cells as available. Total cell number range CO:VOLUMEORAMOUNT_COLLECTED between samples is 6e6 to 20e6 cells per pellet CO:STORAGE_CONDITIONS Pellet CO:STORAGE_VIALS 1.5 mL microfuge tube CO:TISSUE_CELL_IDENTIFICATION YA95 #TREATMENT TR:TREATMENT_SUMMARY Cells were treated with vehicle (dH2O) or 500 ng/mL doxycycline. TR:TREATMENT_COMPOUND Doxycycline TR:TREATMENT_DOSE 500 ng/mL TR:TREATMENT_DOSEVOLUME 4 mL TR:TREATMENT_DOSEDURATION 48 hours TR:TREATMENT_VEHICLE dH2O TR:CELL_GROWTH_CONTAINER 6-well plate #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Amino acid SP:SAMPLEPREP_PROTOCOL_FILENAME SB_AA_Assay.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Amino Acid CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters AccQ Tag (100 x 2mm, 1.7um) CH:METHODS_FILENAME SB_AA_Assay.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Sanford Burnham #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6490 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/mg protein MS_METABOLITE_DATA_START Samples YA95_1 YA95_2 YA95_3 YA95_4 YA95_5 YA95_6 YA95_7 YA95_8 YA95_9 YA95_10 YA95_11 YA95_12 Factors Treatment:Vehicle | Genotype:Odc WT Treatment:Vehicle | Genotype:Odc WT Treatment:Vehicle | Genotype:Odc WT Treatment:Doxycycline | Genotype:Odc WT Treatment:Doxycycline | Genotype:Odc WT Treatment:Doxycycline | Genotype:Odc WT Treatment:Vehicle | Genotype:Odc WT Treatment:Vehicle | Genotype:Odc WT Treatment:Vehicle | Genotype:Odc WT Treatment:Doxycycline | Genotype:Odc -/- Treatment:Doxycycline | Genotype:Odc -/- Treatment:Doxycycline | Genotype:Odc -/- Alanine 2.93 4.30 3.88 4.38 8.93 7.05 3.40 4.24 4.67 4.63 3.62 4.25 Arginine 0.06 0.12 0.07 0.09 0.10 0.13 0.06 0.07 0.09 0.07 0.16 0.07 Asparagine 0.07 0.08 0.07 0.10 0.11 0.12 0.06 0.07 0.10 0.09 0.14 0.10 Aspartic Acid 0.79 0.62 0.74 0.76 0.90 1.11 0.76 0.66 0.77 0.88 1.70 0.90 Citrulline Glutamic Acid 2.77 4.31 2.84 3.46 4.50 7.09 3.55 3.57 4.94 3.86 5.86 3.59 Glutamine 1.98 2.37 2.08 2.22 3.90 4.21 1.78 1.99 2.54 2.39 3.41 2.79 Glycine 1.35 2.94 2.30 3.04 3.25 4.69 2.56 2.83 2.61 2.97 2.45 2.58 Histidine 0.11 0.15 0.08 0.13 0.08 Isoleucine 0.19 0.23 0.21 0.22 0.40 0.37 0.18 0.19 0.27 0.25 0.32 0.25 Leucine 0.16 0.24 0.20 0.19 0.36 0.39 0.14 0.13 0.23 0.26 0.38 0.25 Lysine 0.18 0.17 Ornithine 0.04 0.05 Phenylalanine 0.15 0.19 0.18 0.24 0.35 0.27 0.14 0.16 0.18 0.16 0.24 0.17 Proline 0.73 0.92 0.87 0.94 1.40 1.71 0.99 0.63 1.29 0.63 1.15 0.76 Serine 0.32 0.50 0.37 0.36 0.68 0.77 0.30 0.38 0.55 0.45 0.57 0.44 Threonine 1.10 1.23 1.49 1.36 1.51 1.99 1.09 1.02 1.34 0.90 1.62 1.05 Tryptophan 0.05 0.06 0.09 0.05 0.03 0.05 0.05 Tyrosine 0.13 0.10 0.16 0.19 0.22 0.33 0.13 0.16 0.22 0.15 0.26 0.20 Valine 0.20 0.29 0.23 0.26 0.36 0.52 0.18 0.22 0.32 0.23 0.44 0.26 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID Alanine 4.16 260.17 5950 Arginine 2.68 345.21 6322 Asparagine 2.25 303.13 6267 Aspartic Acid 2.99 304.11 5960 Citrulline 3.33 346.21 9750 Glutamic Acid 3.35 318.11 33032 Glutamine 2.65 317.21 5961 Glycine 2.81 246.08 750 Histidine 2.07 326.21 6274 Isoleucine 8.15 302.21 6306 Leucine 8.26 302.21 6106 Lysine 6.66 487.21 5962 Ornithine 5.93 473.22 6262 Phenylalanine 8.40 336.21 6140 Proline 4.84 286.16 145742 Serine 2.60 276.11 5951 Threonine 3.74 290.11 6288 Tryptophan 8.54 375.21 6305 Tyrosine 6.66 352.21 6057 Valine 7.09 288.21 6287 METABOLITES_END #END