#METABOLOMICS WORKBENCH csun0221_20230406_184959 DATATRACK_ID:3850 STUDY_ID:ST002547 ANALYSIS_ID:AN004194 PROJECT_ID:PR001640 VERSION 1 CREATED_ON April 7, 2023, 10:51 am #PROJECT PR:PROJECT_TITLE LCMS analysis of WT mice treated with PBS or bleomycin PR:PROJECT_SUMMARY C57bl/6 mice were treated with either PBS or bleomycin and waited for 21 days to PR:PROJECT_SUMMARY develop fibrosis. Mouse lungs were resected following by LC-QTOF analysis. PR:INSTITUTE University of Florida PR:LAST_NAME Sun PR:FIRST_NAME Ramon PR:ADDRESS 1200 Newell Drive, ARB, Gainesville, FL, 32610, USA PR:EMAIL ramonsun@ufl.edu PR:PHONE 8594733233 #STUDY ST:STUDY_TITLE C57bl/6 mice subjected to either PBS or bleomycin treatment to develop fibrosis ST:STUDY_SUMMARY C57bl/6 mice subjected to either PBS or bleomycin treatment. By day 21, lung ST:STUDY_SUMMARY fibrosis will develop. We resected the lung and performed LC-QTOF analysis on ST:STUDY_SUMMARY small molecule metabolites between the two groups. ST:INSTITUTE University of Florida ST:LAST_NAME Sun ST:FIRST_NAME Ramon ST:ADDRESS 1200 Newell Drive, ARB, Gainesville, FL, 32610, USA ST:EMAIL ramonsun@ufl.edu ST:PHONE 8594733233 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - WT_Bleo_1 Treatment:Bleomycin Genotype=Wild-type; RAW_FILE_NAME=WT_Bleo_1.d SUBJECT_SAMPLE_FACTORS - WT_Bleo_2 Treatment:Bleomycin Genotype=Wild-type; RAW_FILE_NAME=WT_Bleo_2.d SUBJECT_SAMPLE_FACTORS - WT_Bleo_3 Treatment:Bleomycin Genotype=Wild-type; RAW_FILE_NAME=WT_Bleo_3.d SUBJECT_SAMPLE_FACTORS - WT_Bleo_4 Treatment:Bleomycin Genotype=Wild-type; RAW_FILE_NAME=WT_Bleo_4.d SUBJECT_SAMPLE_FACTORS - WT_Bleo_5 Treatment:Bleomycin Genotype=Wild-type; RAW_FILE_NAME=WT_Bleo_5.d SUBJECT_SAMPLE_FACTORS - WT_PBS_1 Treatment:PBS Genotype=Wild-type; RAW_FILE_NAME=WT_PBS_1.d SUBJECT_SAMPLE_FACTORS - WT_PBS_2 Treatment:PBS Genotype=Wild-type; RAW_FILE_NAME=WT_PBS_2.d SUBJECT_SAMPLE_FACTORS - WT_PBS_3 Treatment:PBS Genotype=Wild-type; RAW_FILE_NAME=WT_PBS_3.d SUBJECT_SAMPLE_FACTORS - WT_PBS_4 Treatment:PBS Genotype=Wild-type; RAW_FILE_NAME=WT_PBS_4.d SUBJECT_SAMPLE_FACTORS - WT_PBS_5 Treatment:PBS Genotype=Wild-type; RAW_FILE_NAME=WT_PBS_5.d #COLLECTION CO:COLLECTION_SUMMARY Approximately 20mg of pulverized, frozen tissue was extracted in 3ml ice-cold CO:COLLECTION_SUMMARY 60% acetonitrile by extensively vortexing. Next, 1ml chloroform was added to CO:COLLECTION_SUMMARY each sample and samples were mixed by manual shaking followed by centrifuging at CO:COLLECTION_SUMMARY 3,200g for 20 minutes at 4°C. The aqueous layer was moved to a new tube and CO:COLLECTION_SUMMARY lyophilized. The middle layer containing protein and insoluble material was CO:COLLECTION_SUMMARY transferred to a new tube, washed twice with 50% methanol, once with 100% CO:COLLECTION_SUMMARY methanol, and briefly dried in a speed-vac. After drying, the insoluble material CO:COLLECTION_SUMMARY was hydrolyzed by heating samples in 3N HCl at 95°C for 2 hours. 100% methanol CO:COLLECTION_SUMMARY was added to hydrolysate to achieve a final concentration of 50% methanol, CO:COLLECTION_SUMMARY samples mixed, centrifuged at 18,000g for 10 min at 4°C, then supernatant dried CO:COLLECTION_SUMMARY on a speed vac. CO:SAMPLE_TYPE Lung CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY mice were treated with either PBS or bleomycin. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Approximately 20mg of pulverized, frozen tissue was extracted in 3ml ice-cold SP:SAMPLEPREP_SUMMARY 60% acetonitrile by extensively vortexing. Next, 1ml chloroform was added to SP:SAMPLEPREP_SUMMARY each sample and samples were mixed by manual shaking followed by centrifuging at SP:SAMPLEPREP_SUMMARY 3,200g for 20 minutes at 4°C. The aqueous layer was moved to a new tube and SP:SAMPLEPREP_SUMMARY lyophilized. The middle layer containing protein and insoluble material was SP:SAMPLEPREP_SUMMARY transferred to a new tube, washed twice with 50% methanol, once with 100% SP:SAMPLEPREP_SUMMARY methanol, and briefly dried in a speed-vac. After drying, the insoluble material SP:SAMPLEPREP_SUMMARY was hydrolyzed by heating samples in 3N HCl at 95°C for 2 hours. 100% methanol SP:SAMPLEPREP_SUMMARY was added to hydrolysate to achieve a final concentration of 50% methanol, SP:SAMPLEPREP_SUMMARY samples mixed, centrifuged at 18,000g for 10 min at 4°C, then supernatant dried SP:SAMPLEPREP_SUMMARY on a speed vac. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Agilent InfinityLab Poroshell 120 EC-C8 (150 x 2.1mm,2.7um) CH:SOLVENT_A 100% water; 10mM ammonium acetate CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 0-15 min linear ramp 90%B to 30%B, 15-18 min isocratic flow of 30%B, 18-19 min CH:FLOW_GRADIENT linear ramp from 30%B to 90%B, and 19-27 min column regeneration with isocratic CH:FLOW_GRADIENT flow of 90%B CH:FLOW_RATE 0.25ml/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Metabolites were measured using an Agilent 6545 quadrapole-time of flight mass MS:MS_COMMENTS spectrometer (MS) coupled to an Agilent 1290 Infinity II UHPLC. Individual MS:MS_COMMENTS samples and standards were acquired with full scan MS in negative mode. Peaks MS:MS_COMMENTS for the deprotonated [M-H]¬- ions were extracted and integrated using Agilent MS:MS_COMMENTS Qualitative Analysis software. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS normalized abundance MS_METABOLITE_DATA_START Samples WT_PBS_1 WT_PBS_2 WT_PBS_3 WT_PBS_4 WT_PBS_5 WT_Bleo_1 WT_Bleo_2 WT_Bleo_3 WT_Bleo_4 WT_Bleo_5 Factors Treatment:PBS Treatment:PBS Treatment:PBS Treatment:PBS Treatment:PBS Treatment:Bleomycin Treatment:Bleomycin Treatment:Bleomycin Treatment:Bleomycin Treatment:Bleomycin Glycogen 0.605233062 0.31144096 0.408441054 0.358180007 0.239911497 0.321150661 0.385355064 0.505263955 0.30419289 0.35147671 alanine 0.0009349 0.00082358 0.00844705 0.00849274 0.00415192 0.00406858 0.00482769 0.00393863 0.00440866 0.00486184 lactate 0.005574986 0.004054798 0.024797797 0.041267525 0.014373707 0.020187162 0.025968448 0.035710483 0.01480433 0.015376958 serine 0.000250325 3.15117E-05 0.000664789 0.000601507 0.00037111 0.001117206 0.001397057 0.001039627 0.000729278 0.001113888 norvaline 0.045042633 0.058210915 0.227861799 0.292377359 0.157563905 0.126897566 0.078398261 0.057810656 0.063060562 0.09888369 succinate 0.000525258 0.000628969 0.002459071 0.002818623 0.001306143 0.001601458 0.002131449 0.001211851 0.001660998 0.001352133 aspartic acid 0.011231498 0.012304176 0.165664304 0.135874554 0.061565885 0.041541047 0.042458234 0.020919815 0.025091175 0.04376086 malate 0.0924343 0.12598456 0.07552393 0.05182775 0.0712876 0.03615668 0.03126342 0.01576377 0.02056699 0.03757182 glutamine 0.0018315 0.00140017 0.0202885 0.01642977 0.00763188 0.01037753 0.01037281 0.00688208 0.00753524 0.01216027 glutamic acid 0.00698358 0.00592375 0.05106516 0.03499098 0.02483869 0.02209001 0.02617074 0.01658751 0.01764384 0.02492585 DHAP/G3P 0 0 0 0.003724501 0 0 0 0 0 0 N-acetyl-aspartate 0.005449591 0.007195353 0.051519539 0.058412113 0.030823235 0.016414528 0.021026921 0.009244828 0.012466844 0.01875474 glucose 0.001148272 0.000700262 0.020883774 0.026216931 0.005215696 0.007498452 0.005029069 0.005162432 0.003272864 0.002507878 3PG 8.89886E-05 0.000135371 0.002588599 0.00066524 0.00194306 0.001378302 0.001021196 0.000482722 0.000716047 0.00060653 citrate 0.1664217 0.12069924 0.16293699 0.119533742 0.13933751 0.0758937 0.04403494 0.03607964 0.03125702 0.05354841 palmitate 0.059327672 0.083714388 0.270704231 0.307199134 0.169930792 0.148506738 0.091935611 0.059359117 0.058633895 0.092315412 G6P 0.028119 0.029534 0.02716506 0.023143 0.025144 0.00404964 0.00232608 0.0032266 0.00127998 0.0017998 F6P 0.0081192 0.029534 0.00708718 0.01221656 0.050005 0.00393262 0.00533279 0.00226718 0.0026718 0.00497691 AMP 0.003138001 0.002901206 0.020115707 0.031979618 0.017307319 0.013097816 0.015601454 0.007321662 0.012195186 0.011795218 ADP 0.00453305 0.001861918 0.00677537 0.006663577 0.03776343 0.01431559 0.009719385 0.002629915 0.006627242 0.004576713 ATP 0.001935485 0.000452254 0.001213456 0.000458244 0.016483115 0.008772914 0.003547585 0.000870076 0.001561953 0.001145418 UDP-glucose 0.0070561 0.0025035 0.0030113 0.00209043 0.0025613 0.00024078 0.000052898 0.000036436 0.00059006 0.000048164 UDP-GlcNAc 0.000128967 0.000120085 0.004558006 0.005419704 0.001053744 0.000734131 0.000256377 0.000314107 0.000330884 0.000341633 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem KEGG Glycogen 439177 C00182 alanine 5950 C00041 lactate 61503 C00186 serine 5951 C00716 norvaline 439575 C01799 succinate 1110 C00042 aspartic acid 5960 C00049 malate 222656 C00149 glutamine 5961 C00064 glutamic acid 33032 C00025 DHAP/G3P 668 C00111 N-acetyl-aspartate 65065 C01042 glucose 5793 C00221 3PG 724 C00597 citrate 311 C00158 palmitate 985 C00249 G6P 5958 C00092 F6P 69507 C00085 AMP 6083 C00020 ADP 6022 C00008 ATP 5957 C00002 UDP-glucose 53477679 C00029 UDP-GlcNAc 9547196 C00043 METABOLITES_END #END