#METABOLOMICS WORKBENCH amitch_20160908_073708 DATATRACK_ID:725 VERSION 1 CREATED_ON November 17, 2016, 3:27 pm #PROJECT PR:PROJECT_TITLE The effect of BCAT1 knockdown on amino acid homeostasis PR:PROJECT_TYPE Amino acid steady-state level analysis PR:PROJECT_SUMMARY Raji cells, a Burkitt lymphoma, expressing a Dox-inducible short hairpin PR:PROJECT_SUMMARY targeting BCAT1 were collected in triplicate at 0, 48, 96, and 144 hours. 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 BCAT1 knockdown on amino acid steady state levels of Raji cells ST:STUDY_TYPE Cells are treated with vehicle or Doxycycline - to induce expression of BCAT1 ST:STUDY_TYPE short hairpin RNA. ST:STUDY_SUMMARY Cells were collected at 0, 48, 96 and 144 hours after Doxycycline addition 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 7 ST:TOTAL_SUBJECTS 21 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 Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS Raji SU:CELL_PRIMARY_IMMORTALIZED Immortalized SU:CELL_PASSAGE_NUMBER 16 SU:CELL_COUNTS 2e6 to 5e6/sample #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_001 Time:- | Treatment:Vehicle SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_002 Time:- | Treatment:Vehicle SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_003 Time:- | Treatment:Vehicle SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_004 Time:48(hrs) | Treatment:Vehicle SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_005 Time:48(hrs) | Treatment:Vehicle SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_006 Time:48(hrs) | Treatment:Vehicle SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_007 Time:48(hrs) | Treatment:Doxycycline SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_008 Time:48(hrs) | Treatment:Doxycycline SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_009 Time:48(hrs) | Treatment:Doxycycline SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_010 Time:96(hrs) | Treatment:Vehicle SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_011 Time:96(hrs) | Treatment:Vehicle SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_012 Time:96(hrs) | Treatment:Vehicle SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_013 Time:96(hrs) | Treatment:Doxycycline SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_014 Time:96(hrs) | Treatment:Doxycycline SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_015 Time:96(hrs) | Treatment:Doxycycline SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_016 Time:144(hrs) | Treatment:Vehicle SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_017 Time:144(hrs) | Treatment:Vehicle SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_018 Time:144(hrs) | Treatment:Vehicle SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_019 Time:144(hrs) | Treatment:Doxycycline SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_020 Time:144(hrs) | Treatment:Doxycycline SUBJECT_SAMPLE_FACTORS Raji_shBCAT1 Raji_021 Time:144(hrs) | Treatment:Doxycycline #COLLECTION CO:COLLECTION_SUMMARY Cells were counted, 2e6 cells were collected via centrifugation, washed twice CO:COLLECTION_SUMMARY with 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 Every 48 hours, up to 144 hours CO:VOLUMEORAMOUNT_COLLECTED 2e6 cells for 0, 48 and 144 time point. 5e6 cells for 96 hour time point CO:STORAGE_CONDITIONS Pellet CO:STORAGE_VIALS 1.5 mL microfuge tube CO:TISSUE_CELL_IDENTIFICATION Raji cells #TREATMENT TR:TREATMENT_SUMMARY Cells were treated with vehicle (dH2O) or 1 ug/mL doxycycline. TR:TREATMENT_COMPOUND Doxycycline TR:TREATMENT_DOSE 1 ug/mL TR:TREATMENT_DOSEVOLUME 10 mL TR:TREATMENT_DOSEDURATION 0, 48, 96, and 144 hours TR:TREATMENT_VEHICLE dH2O TR:CELL_GROWTH_CONTAINER T25 Flask TR:CELL_MEDIA_LASTCHANGED Media changed every two days during time course #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 Raji_001 Raji_002 Raji_003 Raji_004 Raji_005 Raji_006 Raji_007 Raji_008 Raji_009 Raji_010 Raji_011 Raji_012 Raji_013 Raji_014 Raji_015 Raji_016 Raji_017 Raji_018 Raji_019 Raji_020 Raji_021 Factors Time:- | Treatment:Vehicle Time:- | Treatment:Vehicle Time:- | Treatment:Vehicle Time:48(hrs) | Treatment:Vehicle Time:48(hrs) | Treatment:Vehicle Time:48(hrs) | Treatment:Vehicle Time:48(hrs) | Treatment:Doxycycline Time:48(hrs) | Treatment:Doxycycline Time:48(hrs) | Treatment:Doxycycline Time:96(hrs) | Treatment:Vehicle Time:96(hrs) | Treatment:Vehicle Time:96(hrs) | Treatment:Vehicle Time:96(hrs) | Treatment:Doxycycline Time:96(hrs) | Treatment:Doxycycline Time:96(hrs) | Treatment:Doxycycline Time:144(hrs) | Treatment:Vehicle Time:144(hrs) | Treatment:Vehicle Time:144(hrs) | Treatment:Vehicle Time:144(hrs) | Treatment:Doxycycline Time:144(hrs) | Treatment:Doxycycline Time:144(hrs) | Treatment:Doxycycline Alanine 8.61 9.38 10.48 11.99 16.05 30.50 20.10 20.93 N 40.58 21.65 33.49 28.70 41.31 38.33 21.57 13.50 19.53 14.94 21.99 19.71 Arginine 2.84 3.06 3.24 5.56 6.37 17.80 10.56 14.12 N 4.47 4.23 5.79 14.58 25.38 24.73 3.87 4.44 5.30 7.75 8.40 6.74 Asparagine 5.27 5.82 6.22 7.65 8.25 22.21 10.36 14.29 N 16.55 11.74 14.61 14.60 23.72 20.60 14.11 7.58 13.34 13.24 16.64 14.67 Aspartate N N N N N N N N N 1.68 1.47 1.64 1.41 2.16 N 2.06 N 2.18 2.46 3.34 2.48 Citrulline N N 2.68 2.54 2.26 N N 3.33 N 20.13 11.26 14.37 N N N 5.53 4.52 5.79 N N N Glutamate 64.58 68.59 87.80 87.61 83.22 243.36 95.63 163.08 N 94.78 100.75 87.35 133.78 165.10 147.41 102.30 29.68 147.29 129.14 161.97 151.39 Glutamine N N N N N N N N N N N N N N N 39.36 N 50.85 30.62 41.25 28.92 Glycine 9.85 13.34 14.00 18.42 18.42 48.88 22.98 33.27 N 64.83 40.12 46.89 58.87 74.77 67.83 23.51 18.23 18.28 21.51 28.23 27.52 Histidine 0.81 0.95 1.09 1.44 1.71 3.61 1.92 2.28 N 3.53 2.39 3.37 2.93 4.14 4.27 2.00 1.25 1.92 2.38 2.45 2.81 Isoleucine 1.94 2.16 2.78 2.97 4.46 9.92 5.05 8.62 N 12.85 5.84 8.92 8.21 13.29 13.58 5.07 4.35 4.13 5.25 7.75 7.39 Leucine 2.01 2.43 2.51 3.79 4.11 10.21 5.39 6.88 N 10.28 5.95 9.47 9.67 11.16 11.97 4.70 4.60 4.08 5.30 8.05 6.57 Lysine N N N N N N N N N 3.00 2.47 3.23 2.37 4.22 3.93 N N N N N N Ornithine N N N N N N N N N 3.88 2.07 2.43 N N N N 1.91 N N N N Phenylalanine 0.79 0.60 0.93 1.21 1.28 2.69 1.34 2.01 N 3.24 2.34 2.75 2.59 3.54 3.68 1.30 1.49 1.32 1.78 2.38 1.99 Proline 16.13 18.89 21.35 22.58 28.71 49.00 29.29 45.93 N 96.14 64.74 92.85 62.81 91.97 79.31 27.09 34.44 29.56 22.77 26.83 29.85 Serine N N N N N 3.58 2.46 3.98 N 2.15 1.70 1.80 3.64 5.03 4.09 8.47 N 7.19 5.99 9.61 7.59 Threonine N N N N 3.64 7.96 4.27 7.45 N 6.21 4.16 6.09 5.13 8.30 7.29 6.68 N 5.32 6.30 6.94 7.41 Tryptophan N N N N N 1.36 N N N 0.93 0.78 0.94 0.91 1.49 1.24 N N N N N 0.97 Tyrosine N N N N 1.91 4.60 1.95 3.06 N 4.16 3.06 3.98 3.21 6.35 6.22 2.22 N 1.83 2.86 2.94 3.56 Valine N N N N N N N N N 4.12 2.94 3.94 4.03 6.99 5.25 N N N N N N 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 Aspartate 2.99 304.11 5960 Citrulline 3.33 346.21 9750 Glutamate 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