#METABOLOMICS WORKBENCH WXIAOLAB_20230807_080201 DATATRACK_ID:4204 STUDY_ID:ST003492 ANALYSIS_ID:AN005732 PROJECT_ID:PR002141 VERSION 1 CREATED_ON September 4, 2024, 6:01 pm #PROJECT PR:PROJECT_TITLE Secretome of normal primary human pulmonary artery smooth muscle cells PR:PROJECT_SUMMARY This project aims to delineate the possible mediators of aerobic activation of PR:PROJECT_SUMMARY HIF1alpha in normal primary cells and its biological consequences related to PR:PROJECT_SUMMARY pulmonary vascular pathobiology. PR:INSTITUTE Peking University PR:DEPARTMENT Department of Toxicology PR:LABORATORY WXIAO PR:LAST_NAME Xiao PR:FIRST_NAME Wusheng PR:ADDRESS 38 Xueyuan Rd, Haidian District, Beijing, Beijing, 100191, China PR:EMAIL wxiao@bjmu.edu.cn PR:PHONE +1-010-82801628 #STUDY ST:STUDY_TITLE Metabolomic profiling of the secretome in normal primary human pulmonary artery ST:STUDY_TITLE smooth muscle cells ST:STUDY_SUMMARY Growth medium of human pulmonary artery smooth muscle cells (PASMCs) was ST:STUDY_SUMMARY conditioned for 24 hours and analyzed by LC-MS, termed conditioned medium (CM). ST:STUDY_SUMMARY Comparing to fresh complete vascular smooth muscle cell growth medium (GM), ST:STUDY_SUMMARY LC-MS profiling screened 138 metabolites and identified that three branched ST:STUDY_SUMMARY chain alpha-ketoacids, namely KIC, KIV, and KMV are the most abundant ST:STUDY_SUMMARY metabolites in CM of human PASMCs. ST:INSTITUTE Peking University ST:DEPARTMENT Department of Toxicology ST:LAST_NAME Xiao ST:FIRST_NAME Wusheng ST:ADDRESS 38 Xueyuan Rd, Haidian District, Beijing, Beijing, 100191, China ST:EMAIL wxiao@bjmu.edu.cn ST:PHONE +1-010-82801628 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male and female #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 - SmGM 01 Treatment:GM | Sample source:Vascular smooth muscle cells SUBJECT_SAMPLE_FACTORS - SmGM 02 Treatment:GM | Sample source:Vascular smooth muscle cells SUBJECT_SAMPLE_FACTORS - SmGM 03 Treatment:GM | Sample source:Vascular smooth muscle cells SUBJECT_SAMPLE_FACTORS - CMfromSMC 04 Treatment:CM | Sample source:Vascular smooth muscle cells SUBJECT_SAMPLE_FACTORS - CMfromSMC 05 Treatment:CM | Sample source:Vascular smooth muscle cells SUBJECT_SAMPLE_FACTORS - CMfromSMC 06 Treatment:CM | Sample source:Vascular smooth muscle cells #COLLECTION CO:COLLECTION_SUMMARY Human primary pulmonary artery smooth muscle cells were cultured in complete CO:COLLECTION_SUMMARY vascular smooth muscle cell growth medium in a T75 flask. When cells reach to CO:COLLECTION_SUMMARY roungly 80% confluence, growth medium were refreshed with 10 mL and cultured for CO:COLLECTION_SUMMARY 24 hours. Medium samples were then collected and used for LC-MS analysis after CO:COLLECTION_SUMMARY centrifugation and removal of cell debris. CO:SAMPLE_TYPE Vascular smooth muscle cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Human primary pulmonary artery smooth muscle cells were cultured in complete TR:TREATMENT_SUMMARY vascular smooth muscle cell growth medium in a T75 flask. When cells reach to TR:TREATMENT_SUMMARY roungly 80% confluence, growth medium were refreshed with 10 mL and cultured for TR:TREATMENT_SUMMARY 24 hours. Medium samples were then collected (termed conditioned medium, CM) and TR:TREATMENT_SUMMARY used for LC-MS analysis after centrifugation and removal of cell debris. Regular TR:TREATMENT_SUMMARY complete growth medium (GM) of vascular smooth muscle cells was included for TR:TREATMENT_SUMMARY comparison. TR:TREATMENT Conditioned medium (CM) vs regular growth medium (GM) TR:TREATMENT_COMPOUND N/A TR:TREATMENT_ROUTE N/A TR:TREATMENT_DOSE N/A TR:TREATMENT_DOSEVOLUME N/A TR:TREATMENT_DOSEDURATION 24 hours TR:TREATMENT_VEHICLE N/A TR:CELL_GROWTH_CONTAINER 75 cm flasks TR:CELL_GROWTH_CONFIG 1200 rpm TR:CELL_GROWTH_RATE 30-40 hours TR:CELL_MEDIA Complete vascular smooth muscle cell growth medium supplemented with growth TR:CELL_MEDIA factors and 5% FBS TR:CELL_ENVIR_COND 37 C and 5% CO2 TR:CELL_PCT_CONFLUENCE 70-80% TR:CELL_MEDIA_LASTCHANGED Every 48 hours #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites in equal volume of GM and CM were extracted using 100% -80 degree SP:SAMPLEPREP_SUMMARY prechilled LC-MS grade methanol. The final concentration of methanol was 80%. SP:SAMPLEPREP_SUMMARY Proteins were removed by centrifugation and supernatants were collected and SP:SAMPLEPREP_SUMMARY evaporate on an Integrated SpeedVac System to the starting volume (~200 uL). SP:SAMPLEPREP_SUMMARY Twenty microliters of the resulting medium samples were loaded into a LC-MS SP:SAMPLEPREP_SUMMARY sampling tube for analysis. SP:PROCESSING_STORAGE_CONDITIONS Described in summary SP:EXTRACT_STORAGE Described in summary #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Vanquish ultra-high performance liquid chromatography system coupled to a Q CH:CHROMATOGRAPHY_SUMMARY Exactive mass spectrometer CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Merck ZIC-pHILIC(150 mm × 2.1 mm,3.5um) CH:SOLVENT_A 100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT gradient (%B) was 0 min, 80%; 20 min, 20%; 20.5 min, 80%; 28 min, 80%; and 42 CH:FLOW_GRADIENT min CH:FLOW_RATE 100 CH:COLUMN_TEMPERATURE 4 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Focus MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE MALDI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS The mass spectrometer was operated in polarity-switching full scan mode from MS:MS_COMMENTS 70-1000 m/z. Resolution was set to 70,000, and the AGC target was 1×106 ions. MS:MS_COMMENTS Data were acquired and analyzed using TraceFinder 4.1 (Thermo) with peak MS:MS_COMMENTS identifications based on an in-house library of authentic metabolite standards MS:MS_COMMENTS previously analyzed utilizing this method. Missing values were imputed using MS:MS_COMMENTS random forest. Sample peak areas were normalized using probabilistic quotient MS:MS_COMMENTS normalization #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ion intensity MS_METABOLITE_DATA_START Samples SmGM 01 SmGM 02 SmGM 03 CMfromSMC 04 CMfromSMC 05 CMfromSMC 06 Factors Treatment:GM | Sample source:Vascular smooth muscle cells Treatment:GM | Sample source:Vascular smooth muscle cells Treatment:GM | Sample source:Vascular smooth muscle cells Treatment:CM | Sample source:Vascular smooth muscle cells Treatment:CM | Sample source:Vascular smooth muscle cells Treatment:CM | Sample source:Vascular smooth muscle cells 1-methyladenosine 309596 364097 352987 1300373 1351232 1109735 1-methylnicotinamide 307522 388296 227932 229350190 206141298 196868485 2-aminoadipate 994229 987547 1014530 1839290 1144045 1321468 2-deoxycytidine 294471 321107 276174 25014 4730 10963 2-hydroxyglutarate 134580907 135032994 148384566 305626588 322936025 316188938 3-4-dihydroxyphenylglycol 789223 1187993 1238452 862878 796892 863926 3-dehydroxycarnitine 90619 105888 104813 107661 101586 101776 3-hydroxyanthranilic acid 415492 720848 637873 473251 359305 268222 3-methyladipate/pimelate 10656000 11124236 11670043 10872249 10204197 10880349 4-methylcatechol 865252 1330272 1373493 826639 807872 853362 4-pyridoxate 347815 353476 378821 421813 617030 acetylcholine 320976 380247 445500 363420 305272 367719 aconitate 2118659 2327548 2162654 28545583 28367847 30979641 adenine 17239038 18131842 18441123 adenosine 159987 199422 335207 790426 413284 428108 adipate 4613930 3984164 4899247 4356592 4495841 5272868 ADMA 35662009 40903649 43994022 58244908 54896663 60174593 alanine 29257860 29298880 29270329 119122923 91172559 97185580 allantoin 51627641 55395204 53620663 54057522 51923348 55959572 alpha-glycerophosphocholine 21151343 21414440 21918992 25423361 29090630 32538744 alpha-keto-beta-methylvalerate/alpha-ketoisocaproate 195456686 165377962 189672064 alpha-ketoglutarate 6238189 9470511 9577150 15211244 12131640 16118662 alpha-ketoisovalerate 48598817 34753502 43113614 anserine 160643 174304 195773 219741 156274 226213 arginine 1947947082 2136448101 2183804925 1961941162 1819209603 1841241114 ascorbate 1618981 1447495 1292262 1672055 1525846 1872034 asparagine 35874637 43352315 45348419 58707142 48839769 49653585 aspartate 162017459 178807418 175175391 49676518 49472714 48827148 azelate 1518452 1238973 2017153 1476257 1467555 1468628 beta-alanine 1483908 1441173 1702030 2537727 2732576 3177065 betaine 775314673 874584061 900685267 792430608 746379346 788697931 bilirubin 244 302 299 375 441 1099 biotin 36857 43028 44017 37628 35373 38685 butyrate 2618281 2769460 1908502 C14:0 LPC 24439 29223 30200 33470 30836 30993 C16:0 LPC 918507 910079 924215 509970 459507 486017 C16:0 SM 452404 477791 419465 385739 371722 387692 C16:1 LPC 76725 96721 97734 61732 60688 64384 C16:1 SM 24925 29301 24520 25975 23104 24450 C18:0 LPC 81679 83935 76544 33799 29474 28744 C18:0 SM 94069 91946 99275 76804 74384 89098 C18:1 carnitine 103431 89978 79089 38340 24486 37105 C18:2 LPC 44718 66786 69810 8729 10322 8529 C2 carnitine 28641987 33796625 33838275 32203762 32087615 33736794 C20:4 LPC 22230 33731 28852 4426 5112 5555 C22:6 LPC 25606 39479 38876 C3 carnitine 15384097 18521686 18950351 15740096 15704617 14820853 C4 carnitine 18260104 21755491 21903067 19398817 17620127 18552725 C4-OH carnitine 206387 261880 242129 255165 201905 256675 C5 carnitine 5739934 6356662 6655826 5984949 5602204 5917301 C5:1 carnitine 291100 308863 292647 297521 246874 327248 C5-DC carnitine 312591 336398 578877 286042 235658 434330 C6 carnitine 733080 841785 832705 636573 612317 650141 C7 carnitine 107584 110532 118291 113054 96535 112677 carnitine 95320686 106668160 103852007 97840454 89366170 94484276 carnosine 3627869 4228768 4547146 3891196 3519664 3945540 choline 4255141861 4746315423 4669618980 4417875493 4088897371 4041990109 citrate/isocitrate 88026209 89817759 85316731 1062042694 1043689258 1175006557 citrulline 4698777 4642618 4620795 4269732 4132444 4425549 cortisol 3758 4406 4493 4871 4242 4649 creatine 202747169 235453326 288231056 183634480 182232899 228096731 creatinine 838219200 922013164 921120385 829837370 803332906 867577506 cyclohexylamine 20121 18804 28637 21125 19887 26689 cystathionine 94769 70928 83481 83799 112570 cytidine 56402 47730 50832 13629 8219 15033 cytosine 271317 226683 400679 276851 261712 328307 dimethylglycine 1941133 2213854 2049051 2147447 1952601 1945421 DMGV_isomer 1418321 1544331 1709272 1537304 1309742 1540796 eicosapentaenoate 695833 615924 600569 325627 362887 426047 erythronate 6107864 5873685 5290917 4459552 4108228 5983177 fructose/glucose/galactose 1166484693 1177165679 1163788394 724396513 665261641 759054770 fumarate/maleate 47527306 52024134 53961043 86934474 82022792 78474117 glucuronate 3239266 4561969 4598528 6292594 6738360 5138078 glutamate 114441982 129506530 129009922 362243859 344996581 341412017 glutamine 6534159485 7330313965 7317742516 6762710807 6254857629 6474193288 glycerate 2738293 2951195 2962164 3351404 2842404 3035478 glycine 14827849 12464130 12349513 14155250 13453920 14037429 glycodeoxycholate/glycochenodeoxycholate 1630914 1706807 1523210 1336738 1606591 1903006 guanidinoacetic acid 685 714 731 1358 1193 1739 guanine 368 702 2329 9975 13332 11475 guanosine 23 1224 1260 924 hippurate 38956413 46917853 44351661 33876189 33155764 32933916 histidine 1361726560 1482787700 1669819433 1293670690 1224782196 1555375473 homovanillate 455480 428356 548088 505197 585523 541624 hydroxyproline 10190028 11615213 12136954 12845941 11741069 12902890 hypoxanthine 26681559 24498955 25349814 5362370 9738651 13313800 indolelactate 288648 322670 287035 322842 272374 324663 inosine 13862 13979 14970 35357 31377 33557 inositol 288283195 287951506 288797714 303911203 275015686 313490188 isoleucine 1009092278 1097024506 1113194146 912178184 836808765 900310742 isoleucine/leucine 11354769492 11257969644 11410414425 10550711744 9569077955 10755949519 isovalerate 1404400 1366445 1369386 1285465 1203951 1099566 kynurenic acid 3327710 4637874 6359445 3340948 3111131 3814197 lactate 330461024 417170123 386512318 2499861672 2364850673 2592351124 lactose/sucrose/trehalose 1359045 2526822 1920435 824019 1129197 1278954 leucine 1732274992 1948193466 1913549224 1617339044 1462808111 1534855992 lysine 2718233099 3083837625 3025327266 2898680815 2629017777 2539050616 malate 161317542 178587774 180533173 293610558 282387300 269899656 malonate 5404353 5354314 5407229 5366511 5584803 5409525 MDA 614010 4725876 3682163 4181820 methionine 249456089 231939988 225700677 216567187 208802395 234733188 methionine sulfoxide 1649461 1732945 1743018 1738572 1484012 1343168 myristoleic acid 2435 10193 10383 9204 17155 5757 N6-acetyllysine 439443 309934 326479 400359 464329 482953 N-acetylleucine 326 336 610 303 377 328 N-acetylornithine 11273 15332 13587 13222 12129 14193 N-carbamoyl-beta-alanine 146309 176668 167801 5306169 5500319 5282031 niacinamide 2530813204 2929991689 2942142374 2513667767 2334155352 2359365750 nicotinate 525 572 1041 121 195 293 NMMA 7438495 5158976 9073319 12961351 12427318 11858616 ornithine 37456376 33603614 34781958 38435560 30340912 35935595 oxalate 20645610 19273462 16592265 16920557 20928022 20922652 pantothenate 2184513160 2354509748 2406381722 1964797694 1718015244 1743727719 phenylacetate 1108685 7198114 6668544 2546352 2416797 1293643 phenylalanine 909466504 956289305 980653060 883330310 814884385 855884156 phosphocholine 19078 12982 6757 459632 506316 682337 proline 503881556 548268452 533034373 790950593 744504355 743441554 propionate 29204085 31779090 31443159 21876561 20782400 22517480 pterin 461 291 446 2025 2003 2166 pyrocatechol 1325270 1570496 1617298 1283616 1317270 1245689 pyroglutamic acid 5474215990 6133466256 6116172351 5608964883 5195826401 5237398171 pyrrolidonecarboxylic acid 1176632 1253735 1253059 1109715 1029669 1151328 pyruvate 324500832 328719955 309018895 19272417 quinolinate 5228585 6656249 6765507 5921485 5281570 6131700 ribothymidine 1093334 1187361 1118002 6398771 5844250 5963533 salicylurate 1624132 1828733 1791405 1677589 1502317 1642225 SDMA 15369447 15411408 18222597 16789111 16148792 17666457 sebacate 838655 785021 943959 752521 744874 851540 serine 151498618 146718216 145384067 150938959 132697206 142548284 serotonin 1063108 1079143 1309488 904680 858347 1147877 sorbitol 827247482 881183539 880087599 1002520138 927456256 1015209730 sphingosine_isomer 7359 5960 8714 6658 6630 15780 suberate 1435639 1188537 1557081 1206855 1242285 1284952 succinate 494122228 542960289 563012385 544259989 507650506 529853756 taurine 24995887 29551974 30227527 25816638 23300037 24736260 thiamine 888558694 981816440 997192324 924246712 844414172 865685977 threonine 39631675 45307742 46342638 36332973 32621096 32896500 thymine 8428150 8280808 8364441 7630450 6970325 8349606 trimethylamine-N-oxide 7146352 7706329 7419956 7506792 7338787 7143612 tryptophan 98122554 97175811 95759502 79143635 73705716 93349219 tyrosine 79446540 90483539 89821124 78919489 69099570 72087882 uracil 53701178 50652978 51615648 28287804 24687754 30202459 urate 100821126 174378107 182198131 172452781 182754014 115332394 uridine 29211612 31818298 31997591 25583458 33315984 38776896 urocanic acid 178218 96891 129381 173108 91686 107453 valine 4525893674 4558141570 4599656952 4280052193 3981648910 4476911860 xanthine 141293309 153055600 154624289 177998747 191895450 162544722 xanthosine 1103838 1299597 1405826 1614896 1505744 1528403 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 1-methyladenosine 1-methylnicotinamide 2-aminoadipate 2-deoxycytidine 2-hydroxyglutarate 3-4-dihydroxyphenylglycol 3-dehydroxycarnitine 3-hydroxyanthranilic acid 3-methyladipate/pimelate 4-methylcatechol 4-pyridoxate acetylcholine aconitate adenine adenosine adipate ADMA alanine allantoin alpha-glycerophosphocholine alpha-keto-beta-methylvalerate/alpha-ketoisocaproate alpha-ketoglutarate alpha-ketoisovalerate anserine arginine ascorbate asparagine aspartate azelate beta-alanine betaine bilirubin biotin butyrate C14:0 LPC C16:0 LPC C16:0 SM C16:1 LPC C16:1 SM C18:0 LPC C18:0 SM C18:1 carnitine C18:2 LPC C2 carnitine C20:4 LPC C22:6 LPC C3 carnitine C4 carnitine C4-OH carnitine C5 carnitine C5:1 carnitine C5-DC carnitine C6 carnitine C7 carnitine carnitine carnosine choline citrate/isocitrate citrulline cortisol creatine creatinine cyclohexylamine cystathionine cytidine cytosine dimethylglycine DMGV_isomer eicosapentaenoate erythronate fructose/glucose/galactose fumarate/maleate glucuronate glutamate glutamine glycerate glycine glycodeoxycholate/glycochenodeoxycholate guanidinoacetic acid guanine guanosine hippurate histidine homovanillate hydroxyproline hypoxanthine indolelactate inosine inositol isoleucine isoleucine/leucine isovalerate kynurenic acid lactate lactose/sucrose/trehalose leucine lysine malate malonate MDA methionine methionine sulfoxide myristoleic acid N6-acetyllysine N-acetylleucine N-acetylornithine N-carbamoyl-beta-alanine niacinamide nicotinate NMMA ornithine oxalate pantothenate phenylacetate phenylalanine phosphocholine proline propionate pterin pyrocatechol pyroglutamic acid pyrrolidonecarboxylic acid pyruvate quinolinate ribothymidine salicylurate SDMA sebacate serine serotonin sorbitol sphingosine_isomer suberate succinate taurine thiamine threonine thymine trimethylamine-N-oxide tryptophan tyrosine uracil urate uridine urocanic acid valine xanthine xanthosine METABOLITES_END #END