#METABOLOMICS WORKBENCH chenmin_20220814_140231 DATATRACK_ID:3405 STUDY_ID:ST002397 ANALYSIS_ID:AN003904 PROJECT_ID:PR001544 VERSION 1 CREATED_ON December 8, 2022, 11:09 am #PROJECT PR:PROJECT_TITLE Quantify the absolute abundace of metabolite PR:PROJECT_SUMMARY Quantify the absolute abundace of metabolite of yeast under different specific PR:PROJECT_SUMMARY growth rate. PR:INSTITUTE Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University PR:LAST_NAME Chen PR:FIRST_NAME Min PR:ADDRESS State Key Laboratory of Bioreactor Engineering, East China University of Science PR:ADDRESS and Technology PR:EMAIL mchen531@163.com PR:PHONE 18582480786 #STUDY ST:STUDY_TITLE System-level analysis of flux regulation of yeast show that glycolytic flux is ST:STUDY_TITLE controlled by allosteric regulation and enzyme phosphorylation ST:STUDY_SUMMARY Energy metabolism is central for cellular function and has therefore evolved to ST:STUDY_SUMMARY be tightly regulated such that energy production can be balanced to energy ST:STUDY_SUMMARY demand. Energy is being produced in the central carbon metabolism (CCM) and even ST:STUDY_SUMMARY though there has been extensive studies on how fluxes through the different ST:STUDY_SUMMARY pathways in this part of metabolism are regulated. There is little understanding ST:STUDY_SUMMARY of how fluxes are affected by posttranslational modifications and by allosteric ST:STUDY_SUMMARY regulators. Here we integrated multi-omics data (intracellular metabolome, ST:STUDY_SUMMARY extracellular metabolome, proteome, phosphoproteome, and fluxome) under 9 ST:STUDY_SUMMARY different chemostat conditions for building a mathematical model that could map ST:STUDY_SUMMARY functional regulatory events (FREs) in the Saccharomyces cerevisiae. Using ST:STUDY_SUMMARY hierarchical analysis combined with the mathematical model, we observed pathway ST:STUDY_SUMMARY and metabolism-specific flux regulation mechanisms in the CCM. We also found ST:STUDY_SUMMARY that the glycolytic flux increased with specific growth rate, and this increase ST:STUDY_SUMMARY was accompanied by a decrease of both metabolites derived FREs and protein ST:STUDY_SUMMARY phosphorylation level. ST:INSTITUTE Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University ST:LAST_NAME Chen ST:FIRST_NAME Min ST:ADDRESS State Key Laboratory of Bioreactor Engineering, East China University of Science ST:ADDRESS and Technology ST:EMAIL mchen531@163.com ST:PHONE 18582480786 #SUBJECT SU:SUBJECT_TYPE Yeast SU:SUBJECT_SPECIES Saccharomyces cerevisiae SU:TAXONOMY_ID 4932 SU:GENDER Not applicable #FACTORS #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 Wild-type u0.027_pool1_PS Treatment:Control RAW_FILE_NAME=u0.027_pool1_PS.raw SUBJECT_SAMPLE_FACTORS Wild-type u0.051_pool1_PS Treatment:Glucose RAW_FILE_NAME=u0.051_pool1_PS.raw SUBJECT_SAMPLE_FACTORS Wild-type u0.118_pool1_PS Treatment:Glucose RAW_FILE_NAME=u0.118_pool1_PS.raw SUBJECT_SAMPLE_FACTORS Wild-type u0.154_pool1_PS Treatment:Glucose RAW_FILE_NAME=u0.154_pool1_PS.raw SUBJECT_SAMPLE_FACTORS Wild-type u0.207_pool1_PS Treatment:Glucose RAW_FILE_NAME=u0.207_pool1_PS.raw SUBJECT_SAMPLE_FACTORS Wild-type u0.249_pool1_PS Treatment:Glucose RAW_FILE_NAME=u0.249_pool1_PS.raw SUBJECT_SAMPLE_FACTORS Wild-type u0.316_pool1_PS Treatment:Glucose RAW_FILE_NAME=u0.316_pool1_PS.raw SUBJECT_SAMPLE_FACTORS Wild-type u0.355_pool1_PS Treatment:Glucose RAW_FILE_NAME=u0.355_pool1_PS.raw SUBJECT_SAMPLE_FACTORS Wild-type u0.385_pool1_PS Treatment:Glucose RAW_FILE_NAME=u0.385_pool1_PS.raw #COLLECTION CO:COLLECTION_SUMMARY Cells were washed with cold PBS and them flash-frozen in liquid N2 CO:SAMPLE_TYPE Yeast cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY For quantification of intracellular metabolite concentrations,1.0 mL-broth TR:TREATMENT_SUMMARY samples were rapidly withdrawn and quenched in pre-weighed tubes containing 5 mL TR:TREATMENT_SUMMARY cold (−40 °C) pure methanol followed immediately by vigorous vortexing. The TR:TREATMENT_SUMMARY quenched samples were rapidly weighed and poured into a filtration device TR:TREATMENT_SUMMARY containing a cellulose membrane and previously layered with 15 mL of cold TR:TREATMENT_SUMMARY methanol (−40 °C). Subsequently, a vacuum was applied followed by an TR:TREATMENT_SUMMARY immediate additional washing step with 15 mL cold methanol (−40 °C). The TR:TREATMENT_SUMMARY filter containing the cold washed biomass was then transferred into a 50 TR:TREATMENT_SUMMARY mL-falcon tube containing 30 mL of preheated (75 °C) aqueous ethanol solution TR:TREATMENT_SUMMARY (75% v/v). 100 μL of 13C cell extract was added to the tube as an internal TR:TREATMENT_SUMMARY standard. The tube containing the sample was then tightly closed, shaken TR:TREATMENT_SUMMARY vigorously, and placed into a water bath at 95 °C during 3 min for metabolite TR:TREATMENT_SUMMARY extraction. The tubes were then cooled using an ice bath and the filter was TR:TREATMENT_SUMMARY removed. This extract was then concentrated by complete evaporation of the TR:TREATMENT_SUMMARY ethanol–water mixture under vacuum, and resuspended in 500 μL milliQ water. TR:TREATMENT_SUMMARY After a first centrifugation at 15000 g for 5 min at 1 °C, the supernatant was TR:TREATMENT_SUMMARY transferred into a centrifugal filter unit and centrifuged again at the same TR:TREATMENT_SUMMARY conditions. The filtrate was placed into a screw-capped polypropylene vial and TR:TREATMENT_SUMMARY stored at −80 °C until further analysis. Samples were analyzed by LC-MS. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Total liquids are divided into liquid phase vials. SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACT_STORAGE On ice #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters NanoAcquity CH:COLUMN_NAME Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) CH:FLOW_RATE 0.2ml/min CH:COLUMN_TEMPERATURE 40 CH:SOLVENT_A Ammonium formate （5mM) CH:SOLVENT_B 85% Acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo TSQ Quantum Ultra MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Xcalibur #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS umol/l MS_METABOLITE_DATA_START Samples u0.027_pool1_PS u0.051_pool1_PS u0.118_pool1_PS u0.154_pool1_PS u0.207_pool1_PS u0.249_pool1_PS u0.316_pool1_PS u0.355_pool1_PS u0.385_pool1_PS Factors Treatment:Control Treatment:Glucose Treatment:Glucose Treatment:Glucose Treatment:Glucose Treatment:Glucose Treatment:Glucose Treatment:Glucose Treatment:Glucose phosphoenolpyruvic acid 8.618601621 2.232683467 1.120280261 1.760134851 6.713984464 0.768511953 0.347016882 0.968514442 1.019077823 glyceraldehyde 3-phosphate 0.121460635 0.045694607 0.072591789 0.042970954 0.16307067 0.066297482 0.063039568 1.445282221 0.032929685 3-phospho-D-glyceric acid 4.635480583 8.679707733 5.070640609 10.45270648 4.189350549 3.179532337 3.087335884 3.488473162 3.51207178 D-erythrose 4-phosphate 0.093330407 0.0138 0.0101 0.011245 0.109881056 0.009675 0.0085 0.367645649 0.430754257 D-ribose 5-phosphate 0.324964141 0.216170497 0.576211772 0.333079912 0.699808488 1.161806496 0.515096491 0.986549867 1.641584908 α-D-glucose 6-phosphate 4.103195807 8.166494567 11.48597458 7.526147112 10.80558548 19.73160388 9.118207763 9.218333573 9.50267798 fructose 6-phosphate 2.262115348 1.957638298 2.451041802 1.392696194 7.091994652 4.84674652 2.504595383 4.620218219 4.637077431 6-phospho-D-gluconic acid 0.713146304 1.204102396 1.272544054 1.497844732 2.515091761 1.995743245 0.658659938 2.626381483 0.58745655 sedoheptulose 7-phosphate 0.27877432 3.489365414 8.063400301 9.214010696 7.57191164 13.53878418 6.38561305 1.837382897 2.194246412 β-D-fructofuranose 1,6-bisphosphate 0.556389372 1.062707867 1.245841842 1.442486943 1.497032062 2.939011244 2.086310307 1.497297273 0.719646884 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID phosphoenolpyruvic acid 10.26 167.04196 1005 C00074 glyceraldehyde 3-phosphate 5.2 169.05784 729 C00661 3-phospho-D-glyceric acid 9.99 185.0572 439183 D-erythrose 4-phosphate 4.4 199.08382 122357 C00279 D-ribose 5-phosphate 5.19 229.11 439167 C00117 α-D-glucose 6-phosphate 5.15 259.13578 439958 C00092 fructose 6-phosphate 5.17 259.13578 69507 C00085 6-phospho-D-gluconic acid 9.97 275.13518 91493 sedoheptulose 7-phosphate 5.16 289.1618 165007 C05382 β-D-fructofuranose 1,6-bisphosphate 13.5 339.1157 5460765 C00354 METABOLITES_END #END