#METABOLOMICS WORKBENCH JasonMiska_20230206_142517 DATATRACK_ID:3725 STUDY_ID:ST002467 ANALYSIS_ID:AN004023 PROJECT_ID:PR001593 VERSION 1 CREATED_ON February 7, 2023, 8:36 am #PROJECT PR:PROJECT_TITLE Nano-hijacked myeloid cells potentiate antitumor immunity and radiotherapy for PR:PROJECT_TITLE glioblastoma PR:PROJECT_TYPE LC-MS/MS PR:PROJECT_SUMMARY Abstract: Radiation therapy is a key component of the standard of care for PR:PROJECT_SUMMARY glioblastoma (GBM). Although this treatment is known to trigger pro-inflammatory PR:PROJECT_SUMMARY immune responses, it also results in several immune resistance mechanisms such PR:PROJECT_SUMMARY as the upregulation of CD47 by tumors leading to avoidance of phagocytosis and PR:PROJECT_SUMMARY the overexpression of PD-L1 in tumor-associated myeloid cells (TAMCs). PR:PROJECT_SUMMARY Leveraging these RT-elicited processes, we generated a bispecific-lipid PR:PROJECT_SUMMARY nanoparticle (B-LNP) that engaged TAMCs to glioma cells via anti-CD47/PD-L1 PR:PROJECT_SUMMARY dual-ligation. We show that B-LNP blocked these two vital immune checkpoint PR:PROJECT_SUMMARY molecules and promoted the phagocytic activity of TAMCs. In order to boost PR:PROJECT_SUMMARY subsequent T cell recruitment and antitumor activity after tumor engulfment, the PR:PROJECT_SUMMARY B-LNP was encapsulated with diABZI, a non-nucleotidyl agonist for stimulator of PR:PROJECT_SUMMARY interferon genes (STING). In vivo treatment with the diABZI-loaded B-LNP induced PR:PROJECT_SUMMARY a transcriptomic and metabolic switch in TAMCs, transforming them into potent PR:PROJECT_SUMMARY antitumor effector cells, which induced T cell infiltration and activation of in PR:PROJECT_SUMMARY the brain tumors. In preclinical murine glioma models, B-LNP therapy PR:PROJECT_SUMMARY significantly potentiated the antitumor effects of radiotherapy, promoted brain PR:PROJECT_SUMMARY tumor regression, and induced immunological memory against gliomas. The nano37 PR:PROJECT_SUMMARY therapy was efficacious through both intra-tumoral and systemic delivery routes. PR:PROJECT_SUMMARY In summary, our study shows a unique nanotechnology-based approach that hijacks PR:PROJECT_SUMMARY multiple immune checkpoints to boost potent and long-lasting antitumor immunity PR:PROJECT_SUMMARY against GBM. PR:INSTITUTE Northwestern University, Feinberg School of Medicine PR:DEPARTMENT Neurological Surgery PR:LABORATORY Jason Miska PR:LAST_NAME Miska PR:FIRST_NAME Jason PR:ADDRESS 676 N St. Clair PR:EMAIL jason.miska@northwestern.edu PR:PHONE 8478678201 #STUDY ST:STUDY_TITLE Nano-hijacked myeloid cells potentiate antitumor immunity and radiotherapy for ST:STUDY_TITLE glioblastoma ST:STUDY_TYPE IR versus IR + LNP ST:STUDY_SUMMARY Abstract: Radiation therapy is a key component of the standard of care for ST:STUDY_SUMMARY glioblastoma (GBM). Although this treatment is known to trigger pro-inflammatory ST:STUDY_SUMMARY immune responses, it also results in several immune resistance mechanisms such ST:STUDY_SUMMARY as the upregulation of CD47 by tumors leading to avoidance of phagocytosis and ST:STUDY_SUMMARY the overexpression of PD-L1 in tumor-associated myeloid cells (TAMCs). ST:STUDY_SUMMARY Leveraging these RT-elicited processes, we generated a bispecific-lipid ST:STUDY_SUMMARY nanoparticle (B-LNP) that engaged TAMCs to glioma cells via anti-CD47/PD-L1 ST:STUDY_SUMMARY dual-ligation. We show that B-LNP blocked these two vital immune checkpoint ST:STUDY_SUMMARY molecules and promoted the phagocytic activity of TAMCs. In order to boost ST:STUDY_SUMMARY subsequent T cell recruitment and antitumor activity after tumor engulfment, the ST:STUDY_SUMMARY B-LNP was encapsulated with diABZI, a non-nucleotidyl agonist for stimulator of ST:STUDY_SUMMARY interferon genes (STING). In vivo treatment with the diABZI-loaded B-LNP induced ST:STUDY_SUMMARY a transcriptomic and metabolic switch in TAMCs, transforming them into potent ST:STUDY_SUMMARY antitumor effector cells, which induced T cell infiltration and activation of in ST:STUDY_SUMMARY the brain tumors. In preclinical murine glioma models, B-LNP therapy ST:STUDY_SUMMARY significantly potentiated the antitumor effects of radiotherapy, promoted brain ST:STUDY_SUMMARY tumor regression, and induced immunological memory against gliomas. The nano37 ST:STUDY_SUMMARY therapy was efficacious through both intra-tumoral and systemic delivery routes. ST:STUDY_SUMMARY In summary, our study shows a unique nanotechnology-based approach that hijacks ST:STUDY_SUMMARY multiple immune checkpoints to boost potent and long-lasting antitumor immunity ST:STUDY_SUMMARY against GBM. ST:INSTITUTE Northwestern University, Feinberg School of Medicine ST:DEPARTMENT Neurological Surgery ST:LABORATORY Jason Miska ST:LAST_NAME Miska ST:FIRST_NAME Jason ST:ADDRESS 676 N St. Clair ST:EMAIL jason.miska@northwestern.edu ST:PHONE 8478678201 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 6 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - Zha-Jas-20210625-01 Treatment:IR RAW_FILE_NAME=Zha-Jas-20210625-01 SUBJECT_SAMPLE_FACTORS - Zha-Jas-20210625-02 Treatment:IR RAW_FILE_NAME=Zha-Jas-20210625-02 SUBJECT_SAMPLE_FACTORS - Zha-Jas-20210625-03 Treatment:IR RAW_FILE_NAME=Zha-Jas-20210625-03 SUBJECT_SAMPLE_FACTORS - Zha-Jas-20210625-04 Treatment:IR + LNP RAW_FILE_NAME=Zha-Jas-20210625-04 SUBJECT_SAMPLE_FACTORS - Zha-Jas-20210625-05 Treatment:IR + LNP RAW_FILE_NAME=Zha-Jas-20210625-05 SUBJECT_SAMPLE_FACTORS - Zha-Jas-20210625-06 Treatment:IR + LNP RAW_FILE_NAME=Zha-Jas-20210625-06 #COLLECTION CO:COLLECTION_SUMMARY Magnetic bead isolation of cells To isolate specific populations of cells, CO:COLLECTION_SUMMARY single-cell suspensions as isolated above are preblocked with anti-CD16/32 for CO:COLLECTION_SUMMARY 15 min at 4°C. We then used the biotinylated anti-Gr1 (clone RB6-8C5) (all from CO:COLLECTION_SUMMARY Thermo Fisher Scientific) to label murine myeloid cells. Next, the cells were CO:COLLECTION_SUMMARY washed and then incubated with anti-biotin magnetic beads (Miltenyi Biotec) CO:COLLECTION_SUMMARY before performing manual positive selection using MS columns (Miltenyi Biotec). CO:COLLECTION_SUMMARY Purified cells were analyzed for all downstream metabolic analyses. CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY GR1 cells were isolated from mice after 9Gy Radiation or 9Gy radiation and TR:TREATMENT_SUMMARY nanoparticle therapy #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Isolated TAMC and CD8+ T cells samples were dried using a SpeedVac. Acetonitrile SP:SAMPLEPREP_SUMMARY (50%) was added to the tube for reconstitution following overtaxing for 30 s. SP:SAMPLEPREP_SUMMARY Sample solution was then centrifuged for 15 min at 20,000g and 4°C. Supernatant SP:SAMPLEPREP_SUMMARY was collected for LC-MS analysis. The mobile phase A contained 95% water/5% SP:SAMPLEPREP_SUMMARY acetonitrile (v/v), 20 mM ammonium hydroxide, and 20 mM ammonium acetate (pH SP:SAMPLEPREP_SUMMARY 9.0); phase B was 100% acetonitrile. The gradient was performed as follows: 0 SP:SAMPLEPREP_SUMMARY min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1 to 18 min, 75% A; SP:SAMPLEPREP_SUMMARY and 18 to 25 min, 15% A with a flow rate of 400 μl/min. The capillary of the SP:SAMPLEPREP_SUMMARY electrospray ionization source was set to 275°C, with sheath gas at 45 SP:SAMPLEPREP_SUMMARY arbitrary units, auxiliary gas at 5 arbitrary units, and the spray voltage at SP:SAMPLEPREP_SUMMARY 4.0 kV. In positive/negative polarity switching mode, a mass/charge ratio (m/z) SP:SAMPLEPREP_SUMMARY scan range from 70 to 850 was chosen and MS1 data were collected at a resolution SP:SAMPLEPREP_SUMMARY of 70,000. The automatic gain control target was set at 1 × 106, and the SP:SAMPLEPREP_SUMMARY maximum injection time was 200 ms. The top five precursor ions were subsequently SP:SAMPLEPREP_SUMMARY fragmented, in a data-dependent manner, using the higher-energy collisional SP:SAMPLEPREP_SUMMARY dissociation cell set to 30% normalized collision energy in MS2 at a resolution SP:SAMPLEPREP_SUMMARY power of 17,500. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were analyzed by high-performance LC (HPLC) and high-resolution MS and CH:CHROMATOGRAPHY_SUMMARY MS/MS (HPLC-MS/MS). The system consists of Thermo Q Exactive with an CH:CHROMATOGRAPHY_SUMMARY electrospray source and an UltiMate3000 (Thermo Fisher Scientific) series HPLC CH:CHROMATOGRAPHY_SUMMARY consisting of a binary pump, degasser, and autosampler outfitted with an XBridge CH:CHROMATOGRAPHY_SUMMARY Amide column (Waters; dimensions of 4.6 mm by 100 mm and a 3.5-μm particle CH:CHROMATOGRAPHY_SUMMARY size). CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer CH:COLUMN_NAME Water's Xbridge amide (100 x 3mm, 3.5 um) CH:SOLVENT_A 95% water/5% acetonitrile; 20 mM ammonium hydroxide; 20 mM ammonium acetate CH:SOLVENT_A (pH 9.0) CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1 to 18 min, 75% CH:FLOW_GRADIENT A; and 18 to 25 min, 15% A CH:FLOW_RATE 400 μl/min CH:COLUMN_TEMPERATURE 275 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS Isolated TAMC and CD8+ T cells samples were dried using a SpeedVac. Acetonitrile MS:MS_COMMENTS (50%) was added to the tube for reconstitution following overtaxing for 30 s. MS:MS_COMMENTS Sample solution was then centrifuged for 15 min at 20,000g and 4°C. Supernatant MS:MS_COMMENTS was collected for LC-MS analysis. The mobile phase A contained 95% water/5% MS:MS_COMMENTS acetonitrile (v/v), 20 mM ammonium hydroxide, and 20 mM ammonium acetate (pH MS:MS_COMMENTS 9.0); phase B was 100% acetonitrile. The gradient was performed as follows: 0 MS:MS_COMMENTS min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1 to 18 min, 75% A; MS:MS_COMMENTS and 18 to 25 min, 15% A with a flow rate of 400 μl/min. The capillary of the MS:MS_COMMENTS electrospray ionization source was set to 275°C, with sheath gas at 45 MS:MS_COMMENTS arbitrary units, auxiliary gas at 5 arbitrary units, and the spray voltage at MS:MS_COMMENTS 4.0 kV. In positive/negative polarity switching mode, a mass/charge ratio (m/z) MS:MS_COMMENTS scan range from 70 to 850 was chosen and MS1 data were collected at a resolution MS:MS_COMMENTS of 70,000. The automatic gain control target was set at 1 × 106, and the MS:MS_COMMENTS maximum injection time was 200 ms. The top five precursor ions were subsequently MS:MS_COMMENTS fragmented, in a data-dependent manner, using the higher-energy collisional MS:MS_COMMENTS dissociation cell set to 30% normalized collision energy in MS2 at a resolution MS:MS_COMMENTS power of 17,500. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Normalized peak area MS_METABOLITE_DATA_START Samples Zha-Jas-20210625-01 Zha-Jas-20210625-02 Zha-Jas-20210625-03 Zha-Jas-20210625-04 Zha-Jas-20210625-05 Zha-Jas-20210625-06 Factors Treatment:IR Treatment:IR Treatment:IR Treatment:IR + LNP Treatment:IR + LNP Treatment:IR + LNP (S)-4-hydroxymandelonitrile 272476 187098 148565 81421 712900 434657 1,3-bisphospho-D-glycerate 6263917 9058076 8058918 2069914 13117887 3788895 1-methylnicotinamide+ 0 0 0 2792617 17617170 14958269 1-phenylethylamine 6454873 3587045 2617278 2786468 3368768 2377033 2- / 3-phosphoglycerate 38736246 44890450 34332090 44579378 62659596 39837433 2-(Formamido)-N1-(5-phospho-D-ribosyl)acetamidine 0 0 0 0 0 0 2,3-bisphospho-D-glycerate 0 0 0 0 0 0 2-aminobutanoate / 2-aminoisobutanoate 14566490 71386144 12820411 9739895 9614177 10110497 2-HG 1707088 498898 830454 786590 441963 264808 2-hydroxybutanoic acid 1830653 2283394 1820562 2403435 2284568 2109845 2-keto-isovaleric acid 3322779 6276340 5691331 8134843 8533267 5697467 2-ketovaleric acid 0 0 0 0 0 0 2-oxoadipic acid 299991 311175 465596 384353 186531 0 2-oxobutanoic acid 9384638 23157730 21338340 25486763 56136063 55495926 3- / 4 -hydroxybutanoic acid 47943599 42574046 37179110 48796756 54360462 65489890 3- / 4-hydroxyphenylacetic acid / vanillin / 6836661 6678657 7072598 5876842 5150301 4562461 3- / 7-methylguanine 471722 454629 313972 52868 182320 76663 3,4-dihydroxymandelic acid 0 0 0 0 0 0 3,4-dihydroxyphenylpropanoate 12903 27292 24054 0 15341 11280 3-aminoisobutanoate 0 0 0 0 0 0 3-hydroxy-anthranilic acid 494582 203194 249458 206035 180329 248458 3-hydroxykynurenine 0 0 0 0 0 0 3-hydroxypropanoic acid 0 0 0 0 0 0 3-methoxytyramine 0 0 0 0 0 0 3-phosphohydroxypyruvic acid 0 185362 0 0 0 0 3-phospho-serine 8286866 7985580 8815299 13502454 12587523 9999077 3-ureidopropionic acid 429041 69209 122092 465426 1444790 1076560 4-hydroxyphenylpyruvic acid 0 0 0 0 24519 0 4-hydroxyproline / 5-aminolevulinate / N-acetyl-b-alanine 14254238 13186677 16235269 4219744 2448221 1402869 4-phosphopantothenate 0 0 0 0 0 0 4-trimethylammoniobutanoate+ 15093882 9034312 9632315 6042373 5213319 2689892 5,6-dihydroxyindole 0 0 0 0 0 0 5-HIAA 15938 0 10417 28848 9876 0 5-methylcytosine 2428204 1286670 727951 1257153 1571172 746473 5-methyl-THF 0 0 0 0 0 0 5'-methylthioadenosine 35416995 33268361 35520454 25104051 22827526 16426689 6-aminocaproic acid 6739697 11738147 23454759 7165117 12416428 14426422 6-methylnicotinamide 93918527 110773023 53501161 22247906 10000398 8264686 6-O-methylguanine 0 0 0 0 0 0 6-phosphogluconic acid 67139 166112 35807 54529 49439 105135 6-phosphoglucono-D-lactone 0 0 0 0 0 0 acetoacetic acid / succinic semialdehyde 29571699 28678489 26187208 32078982 16995021 10661179 acetylcholine+ 84040616 66960674 59614939 26383199 11328997 12039465 acetyl-CoA 0 0 0 0 0 0 aconitic acid 6656221 25681855 38493755 23978694 20899336 16714572 adenine 19216590 15994680 20915598 8173456 15656449 19471924 adenosine 1284376 1028337 1718529 553715 3355708 1466319 adenylosuccinate 9215109 7690237 8274386 5974290 10562991 9258364 ADP / dGDP 102602960 110345820 72830535 338898462 102774987 85231337 ADP-D-glucose 0 0 0 0 0 0 agmatine 74394 89001 22007 12194 0 0 AICA ribonucleotide 65964 20821 17730 432642 261610 124814 a-KG 577056 539165 0 614238 109110 0 alanine 237110846 244555350 245092509 162079912 82661385 39370735 alanine methyl ester 7394491 5768674 7138350 2632657 6671296 3075806 allantoin 2889097 2479564 2150554 1905082 1841476 1249719 AMP / dGMP 729175265 852190381 711302230 399179355 190322864 101313005 anandamide 0 0 0 0 0 0 arginine 1106480911 864343074 1056771400 990658630 880968694 596527677 ascorbic acid 1583133 660330 849157 465099 418393 29693 asparagine 16046774 15649378 13411039 14825615 13537101 8902243 aspartic acid 101904523 95906249 92956926 97905108 89018034 66321178 ATP / dGTP 4117067 3640995 2226037 49549806 5313389 4736175 b-alanine 8167663 13328826 9307891 4799629 2295036 2265819 betaine 2208577262 2155221016 2147777167 1788328234 1991098421 1845531690 butyric acid 262769 419939 119354 722146 553924 132562 butyric acid(iso) 754571 1428271 954845 973096 553924 450375 carbamoyl phosphate 55695 67281 29741 39330 0 55516 carnitine+ 88225821 83629993 153494253 101213436 45565551 24367367 CDP 6629768 7020073 5105447 13268000 5603093 4475969 CDP-choline 15580234 13747479 12327789 9645357 5477157 3365592 cGMP 80119 19173 61579 0 93384 0 choline+ 6313478386 5669654126 5303238771 3683307920 5937171366 4458577504 citraconic acid 18718374 16966134 22900021 21341408 16413648 12325938 citramalic acid 13648198 11599135 9991747 12466489 10065500 10970110 citrate / citrate(iso) 3151936559 3287274347 3524203697 3466672061 3456134107 3155493346 citrulline 0 0 0 880685 886413 206726 CMP 89932685 97849219 86672364 32485956 62457550 38281089 cortisol 0 129373 0 179859 26816 0 creatine 579827342 492129686 644400170 673790406 240199497 110988477 creatinine 10039249 11506583 14757286 6732093 11308145 12841749 CTP 233174 274651 37762 3827681 412846 307711 cystathionine 7372846 10127905 5779815 27470862 23165329 10130585 cysteine 5380761 3914343 4105396 3247616 4230692 1724936 cysteine-S-sulfate 264413 104925 52249 142827 192104 198670 cystine 6890630 7245762 4350513 9206038 4633902 2809047 cytidine 26048487 23005203 18540589 8515916 21847518 8593080 cytosine 7293024 5592076 3769155 7575582 6442169 3406182 dADP 0 0 0 0 0 0 dAMP 0 0 0 0 0 0 D-arabinono-1,4-lactone 58636266 49626200 67286799 26513712 8357814 2964526 dATP 0 0 0 0 0 0 dCDP 432103 542567 391132 666131 0 0 dCMP 2457014 2000414 1872492 1700380 2213858 1238151 dCTP 39052 100915 36517 486367 197200 98349 dehydroascorbic acid 138543536 76985371 86921341 68723056 71208941 38907569 deoxyguanosine 0 0 0 0 0 0 deoxyinosine 292259 192566 106474 43608 105619 11614 deoxyuridine 99950 45262 101905 11690 12595 10840 D-erythrose-4-phosphate 0 15042 0 16469 0 0 D-galactonic acid / D-gluconic acid 17382131 15926184 15123461 6401078 6831735 3488682 D-galactosamine/glucosamine-1/6-phosphate 410720 789218 549135 2868524 2512708 1941106 dihydroorotate 40336 0 0 20295 0 0 dimethylglycine 0 0 0 0 0 0 DL-DOPA 10481 0 0 0 8843 0 DOPAC 31439 36981 37231 61321 43531 0 dopamine 0 0 0 0 0 0 D-ribose-1/5-phosphate / D-ribulose 5-phosphate 57476979 54803869 41692835 12802677 44948294 25692308 D-sedoheptulose-7-phosphate 4571886 5860668 5093408 7297139 7732902 5369107 dTMP 0 0 30124 0 0 0 dTTP 0 0 0 0 0 0 dUMP 0 0 0 0 0 0 dUTP 0 0 0 0 0 0 epinephrine 384062 260472 401036 438436 518948 451229 ethyl acetate 2293804 2266660 2360790 2262993 2806907 3196119 F-1,6/2,6-DP 4611398 5678283 5315982 13311967 10414444 9061458 F1P 352885 471579 311728 820133 626893 311728 F6P / G1P / G6P 10664360 14294304 10415287 30153741 33327552 21418649 FAD 0 0 0 0 0 0 FMN 0 0 0 0 0 0 folic acid 0 0 0 0 0 0 fructose / mannose 1950457 397309 5173610 3013045 76455 3009928 fumaric acid 1011094 1518434 1502434 880842 990693 170386 GABA 3931787 2750473 2920565 8925297 6417260 4773502 galactosamine / glucosamine 56467 38222 120982 105187 19995 43825 galactose / glucose 6777018 7093005 9699105 3227665 17722431 3952959 GalNAC/GlcNAC/ManNAC 4534625 4675316 5491114 8264012 8224373 6825355 GAP / DHAP 2665146 2366792 2118754 5417253 7156220 1630562 GDP 20339883 24446535 16911945 62376219 30743790 25638997 GDP-L-fucose 2627511 2778283 2052763 1958493 2073432 1384702 glucono-lactone 6641721 3652944 4411963 3614952 4657731 2343594 glutaconic acid 247338331 164881310 167777857 142103753 116175634 57605039 glutamine 38367552 35729762 33915531 15660031 12579233 7316984 glutaminic acid 96354909 87077348 75626419 92134699 88692415 63718704 glutathione 7769926 4162202 4929224 2713647 4005118 1875709 glutathione disulfide 163746058 180522917 136587517 156923806 112734443 94618030 glyceraldehyde 0 0 0 0 0 0 glyceric acid 29145035 26121632 22060322 35979439 47453585 59810557 glycerol 2/3-phosphate 21415291 18315756 15716462 28286372 21700969 13196437 glycine 47489735 146449732 40280823 23636307 16692441 8681113 glycylglycine 315796 447508 535914 0 0 0 GMP 225488198 222158002 170187856 68790496 156979552 101636223 GTP 874082 905431 372134 8805784 1909849 3230415 guanidine 392048 2390251 1055173 2268869 303812 326899 guanidinoacetate 15059410 14596236 23176260 4215135 925059 424190 guanine 1415745 924157 890587 99140 287219 135800 guanosine 5901578 5574923 3832492 475789 1336335 634939 gulonic-lactone 3897123 1758603 2770615 3454226 2773953 2452050 histamine 2500460 2135430 1499555 14294666 8086716 3349138 histidine 222616060 226948647 195701588 129969923 77050220 43936501 homocysteine 0 0 0 0 0 0 homoserine / theronine 111384758 119840860 110001729 71886129 38199321 23628561 hydroxyphenyllactic acid / HVA 2582501 2144551 2074261 1295140 1644337 1154075 hypoxanthine 384006102 350588772 265905542 23433759 221503175 105391802 IDA 270225 293074 245931 327618 1607339 683468 IDP 47823579 23745149 13053357 39227699 96407556 97113638 imidazole-4-acetate 883107 544167 686125 750863 564593 402016 IMP 47451005 56831311 47359605 58410665 47508725 36114594 inosine 49272647 38228726 36025507 1586014 16439251 7912510 isonictinic acid 1317210 914081 1270161 372751 717992 469766 itaconic acid 306872085 537749404 559747656 271585618 9560085 4341191 kynurenic acid 0 7774 7338 0 8855 0 kynurenine 1586014 2788717 1531898 3863904 3244323 2484480 lactic acid 10426953298 9415510872 8347881177 12851676707 19735038273 19731483750 L-arginino-succinate 5520579 4002428 4533738 32303938 29611726 16625195 L-cysteic acid 0 0 0 0 0 0 leucine / leucine(iso) 1930079906 1822484411 1699573853 1048739698 1036996109 686797272 levulinic acid 0 0 0 0 0 0 L-NMMA 14463776 13224799 10368205 4804524 4936608 2764565 L-tyrosine methyl ester 0 0 0 0 0 0 lysine 1441452361 1396837413 1027635418 743556570 727309365 463392642 maleic acid 0 0 0 0 0 0 malic acid 167885822 177673414 206443141 130843784 46599640 32168690 malonyl-CoA 0 0 0 0 0 0 mesaconic acid 7533112 6715811 5969821 0 0 0 methionine 612082075 559051009 475479119 239683004 328709674 215620375 methyl acetoacetate 0 3356391 8586256 5190881 0 0 methylglyoxal 1070320 0 0 1338159 0 0 methylmalonic acid 21724041 10929154 20911411 15424999 5127082 0 mevalonic acid 13824957 9444656 8982791 21060737 24160816 26632460 N1/N8-acetylspermidine 2568921 113453718 159917315 55922098 68319995 97084514 N-acetylaspartic acid 19875670 14721114 15128013 21031425 16712246 11626577 N-acetylaspartylglutamic acid 2237680 1821347 2420103 3895066 2244567 1449064 N-acetyl-DL-alanine 116149604 107041442 84639367 65122678 94129810 83893854 N-acetyl-DL-serine 203166460 129048201 112224583 53850518 43345646 31745184 N-acetyl-dopamine 18552 31835 23905 30418 48519 40021 N-acetylglucosamine 1/6-phosphate 3933039 3534177 2476253 5286544 5123771 3467993 N-acetyl-L-glutamic acid 141065 188180 125872 0 0 0 NAD+ 23623288 26676872 23252805 21350538 2224048 4967012 NADH 0 0 0 0 0 0 NADP+ 4798811 5732618 4650292 13792573 586628 1809339 NADPH 0 0 6802 6819 0 0 N-carbamoyl-L-aspartate 0 0 0 0 0 0 nebularine 292142 192573 202888 43675 315238 210147 nicotinamide 655147338 795914963 641449014 371048271 717157760 444812583 nicotinamide riboside+ 0 0 0 0 0 0 nicotinic acid 5266509 4778213 4016307 2622394 3303589 3001314 N-methyltryptamine 65247 15358 11073 22752 0 32481 NMN 2380316 2964245 1909481 3074566 2265657 1607514 norepinephrine 0 42126 49051 29757 56441 47473 O-acetyl-L-serine 6263579 129048201 112224583 53850518 20407587 17681138 octopamine 983506 848077 1102805 680009 802346 950922 ornithine 650003388 697759945 491360946 214767701 218480810 114124552 orotic acid 71746 0 0 351251 0 0 orotidylic acid 0 0 0 18257 0 0 oxalacetic acid 0 97706 92874 0 0 0 pantothenic acid 9352602 7516423 6413157 21017251 14688076 6730017 phenethylamine 0 0 0 0 0 0 phenylalanine 297845433 299525247 332222788 83676826 87111785 55651802 phenylephrine / synephrine 225450 242570 253557 45528 174783 157095 phosphocreatine 59121521 68857639 44002590 345297276 191435925 129134984 phosphoenolpyruvate 33101936 43600527 38072088 10021209 68646385 19648489 phosphorylcholine+ 1567319441 1491394318 1326566654 595837577 782271237 446806668 picolinic acid 5346627 5812676 5263509 5895036 8274244 9452075 proline 2110565514 1947914443 1762952356 1721177186 1756831280 1365362622 propionic acid 0 0 0 0 0 0 PRPP 0 0 11496 0 0 0 pseudouridine 673965 720888 509026 729016 448111 434130 putrescine 8982551 55433431 49536459 35095006 23114607 42040294 pyridoxine 279969 173366 249628 497097 302469 288100 pyroglutamic acid 109220224 93591548 89598171 87003059 266192711 251742127 pyrroline hydroxycarboxylic acid 9181363 8317844 7629821 3648397 2330997 0 pyruvic acid 59936405 62466489 57634377 138048936 76557227 75635927 quinolinic acid 3592726 9300153 4668111 3027269 1348003 410732 S-adenosyl-L-homocysteine 0 0 0 0 0 0 S-adenosyl-L-methionine+ 48126758 38048544 34731000 36702411 35967324 21812175 S-adenosylmethioninamine+ 0 0 0 0 0 0 SAICAR 0 0 6748 54317 41946 34527 sarcosine 7352201 14668623 14065758 9585958 29972342 27588050 serine 27077301 25439153 23200362 16106999 16938240 9974749 serotonin 0 0 0 152600 0 0 sorbitol / galactitol / L-iditol / manitol 2240077 2176999 2376181 2485624 2463661 1817621 spermidine 401705068 344525401 294037681 174197026 108679001 104871668 spermine 93866507 105829843 109946603 72389167 54300178 57517523 succinic acid 7269362 8677819 7841477 8254050 6443790 4124423 succinyl-CoA 14531 9392 12844 0 0 18206 taurine 3709105657 3092774208 3118334070 2832791030 2895797567 2104018744 thiamine+ 13349372 12375439 18651215 27145036 31727883 21265418 thymidine 3925780 3014356 2239169 3718423 2635635 1631379 thymine 2401737 3917809 1877444 4245395 6871721 4080461 tryptamine 161559 60140 57917 15376 78346 71375 tryptophan 37831464 40728994 44299427 14993681 14157729 8862800 tyramine 1033129 1208197 707287 626903 936134 469206 tyrosine 231938165 216314552 176362997 102299716 144102764 88632397 UDP 7233895 8430661 5103095 14331004 6011760 6040271 UDP-D-glucose 2640272 3789237 2032571 3496185 2536714 1978208 UDP-N-acetyl-galactosamine/UDP-N-acetyl-glucosamine 14293299 14491778 11640963 9065105 8338058 5015676 UMP 48757063 64008528 38037086 19415324 31826214 22456762 uracil 17339706 29605543 17608205 14004452 23429856 14214893 urea 2119067 2155264 1961761 849292 916945 742527 uric acid 35928813 46245140 34989838 51860680 54843091 41368970 uridine 3996814 4399580 3456420 701271 1329961 1016888 UTP 106780 364240 75140 2307028 237610 257456 valine 301539751 303901357 233863237 272319398 282505295 221495368 xanthine 20722210 19488025 22561951 2026289 13524106 6471120 xanthosine 532847 578308 389517 648636 992868 484488 xanthurenic acid 0 0 0 0 0 0 XMP 6551073 7800390 5536429 5535523 4181358 2777879 y-glutamylcysteine 0 0 0 0 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID (S)-4-hydroxymandelonitrile C03742 1,3-bisphospho-D-glycerate C00236 1-methylnicotinamide+ C02918 1-phenylethylamine C02455 2- / 3-phosphoglycerate C00631 / C00197 2-(Formamido)-N1-(5-phospho-D-ribosyl)acetamidine C04640 2,3-bisphospho-D-glycerate C01159-C03339 2-aminobutanoate / 2-aminoisobutanoate C02261 / C03665 2-HG C02630 2-hydroxybutanoic acid C05984 2-keto-isovaleric acid C00141 2-ketovaleric acid C06255 2-oxoadipic acid C00322 2-oxobutanoic acid C00109 3- / 4 -hydroxybutanoic acid C01089 / C00989 3- / 4-hydroxyphenylacetic acid / vanillin / C05593 / C00642 / C00755 3- / 7-methylguanine C02230 / C02242 3,4-dihydroxymandelic acid C05580 3,4-dihydroxyphenylpropanoate C10447 3-aminoisobutanoate C05145 3-hydroxy-anthranilic acid C00632 3-hydroxykynurenine C02794 3-hydroxypropanoic acid C01013 3-methoxytyramine C05587 3-phosphohydroxypyruvic acid C03232 3-phospho-serine C01005 3-ureidopropionic acid C02642 4-hydroxyphenylpyruvic acid C01179 4-hydroxyproline / 5-aminolevulinate / N-acetyl-b-alanine C01157 / C00430 / C01073 4-phosphopantothenate C03492 4-trimethylammoniobutanoate+ C01181 5,6-dihydroxyindole C05578 5-HIAA C05635 5-methylcytosine C02376 5-methyl-THF C00440 5'-methylthioadenosine C00170 6-aminocaproic acid C02378 6-methylnicotinamide n/a 6-O-methylguanine n/a 6-phosphogluconic acid C00345 6-phosphoglucono-D-lactone C01236 acetoacetic acid / succinic semialdehyde C00164 / C00232 acetylcholine+ C01996 acetyl-CoA C00024 aconitic acid C00417-C02341 adenine C00147 adenosine C00212 adenylosuccinate C03794 ADP / dGDP C00008 / C00361 ADP-D-glucose C00498 agmatine C00179 AICA ribonucleotide C04677 a-KG C00026 alanine C00041-C00133 alanine methyl ester n/a allantoin C01551 AMP / dGMP C00020 / C00362 anandamide C11695 arginine C02385 ascorbic acid C00072 asparagine C16438 aspartic acid C16433 ATP / dGTP C00002 / C00286 b-alanine C00099 betaine C00719 butyric acid C00246 butyric acid(iso) C02632 carbamoyl phosphate C00169 carnitine+ C00487 CDP C00112 CDP-choline C00307 cGMP C00943 choline+ C00114 citraconic acid C02226 citramalic acid C00815 citrate / citrate(iso) C00158 / C00311 citrulline C00327 CMP C00055 cortisol C00735 creatine C00300 creatinine C00791 CTP C00063 cystathionine C00542 cysteine C00736-C00097 cysteine-S-sulfate C05824 cystine C01420 cytidine C00475 cytosine C00380 dADP C00206 dAMP C00360 D-arabinono-1,4-lactone C00652 dATP C00131 dCDP C00705 dCMP C00239 dCTP C00458 dehydroascorbic acid C05422 deoxyguanosine C00330 deoxyinosine C05512 deoxyuridine C00526 D-erythrose-4-phosphate C00279 D-galactonic acid / D-gluconic acid C00880 / C00257 D-galactosamine/glucosamine-1/6-phosphate C03783 / C00352 / C06377 / C06156 dihydroorotate C00337 dimethylglycine C01026 DL-DOPA C00355 DOPAC C01161 dopamine C03758 D-ribose-1/5-phosphate / D-ribulose 5-phosphate C00620 / C00117 / C00199 D-sedoheptulose-7-phosphate C05382 dTMP C00364 dTTP C00459 dUMP C00365 dUTP C00460 epinephrine C00788 ethyl acetate C00849 F-1,6/2,6-DP C00354 / C00665 F1P C01094 F6P / G1P / G6P C00085 / C00103 / C00092 FAD C00016 FMN C00061 folic acid C00504 fructose / mannose C00095 / C00159 fumaric acid C00122 GABA C00334 galactosamine / glucosamine C02262 / C00329 galactose / glucose C00124 / C00267 GalNAC/GlcNAC/ManNAC C01074 / C00140 / C00645 GAP / DHAP C00118 / C00111 GDP C00035 GDP-L-fucose C00325 glucono-lactone C00198 glutaconic acid C02214 glutamine C00303 glutaminic acid C00302 glutathione C00051 glutathione disulfide C00127 glyceraldehyde C02426 glyceric acid C00258 glycerol 2/3-phosphate C02979 / C00093 glycine C00037 glycylglycine C02037 GMP C00144 GTP C00044 guanidine C17349 guanidinoacetate C00581 guanine C00242 guanosine C00387 gulonic-lactone C01040 histamine C00388 histidine C00768 homocysteine C05330 homoserine / theronine C00263 / C00188 hydroxyphenyllactic acid / HVA C03672 / C05582 hypoxanthine C00262 IDA C19911 IDP C00104 imidazole-4-acetate C02835 IMP C00130 inosine C00294 isonictinic acid C6H5NO2 itaconic acid C00490 kynurenic acid C01717 kynurenine C01718 lactic acid C01432 L-arginino-succinate C03406 L-cysteic acid C00506 leucine / leucine(iso) C16439 / C16434 levulinic acid n/a L-NMMA C03884 L-tyrosine methyl ester C03404 lysine C00047/C00739 maleic acid C01384 malic acid C00711 malonyl-CoA C00083 mesaconic acid C01732 methionine C00073 methyl acetoacetate n/a methylglyoxal C00546 methylmalonic acid C02170 mevalonic acid C00418 N1/N8-acetylspermidine C00612 / C01029 N-acetylaspartic acid C01042 N-acetylaspartylglutamic acid C12270 N-acetyl-DL-alanine n/a N-acetyl-DL-serine n/a N-acetyl-dopamine n/a N-acetylglucosamine 1/6-phosphate C04501 / C00357 N-acetyl-L-glutamic acid C00624 NAD+ C00003 NADH C00004 NADP+ C00006 NADPH C00005 N-carbamoyl-L-aspartate C00438 nebularine C01736 nicotinamide C00153 nicotinamide riboside+ C03150 nicotinic acid C00253 N-methyltryptamine C06213 NMN C00455 norepinephrine C00547 O-acetyl-L-serine C00979 octopamine C04227 ornithine C01602 orotic acid C00295 orotidylic acid C01103 oxalacetic acid C00036 pantothenic acid C00864 phenethylamine C05332 phenylalanine C02057 phenylephrine / synephrine C07441 / C04548 phosphocreatine C02305 phosphoenolpyruvate C00074 phosphorylcholine+ C00588 picolinic acid C10164 proline C16435 propionic acid C00163 PRPP C00119 pseudouridine C02067 putrescine C00134 pyridoxine C00314 pyroglutamic acid C01879-C02237 pyrroline hydroxycarboxylic acid C04281 pyruvic acid C00022 quinolinic acid C03722 S-adenosyl-L-homocysteine C00021 S-adenosyl-L-methionine+ C00019 S-adenosylmethioninamine+ C01137 SAICAR C04823 sarcosine C00213 serine C00716 serotonin C00780 sorbitol / galactitol / L-iditol / manitol C00794 / C01697 / C01507 / C00392 spermidine C00315 spermine C00750 succinic acid C00042 succinyl-CoA C00091 taurine C00245 thiamine+ C00378 thymidine C00214 thymine C00178 tryptamine C00398 tryptophan C00806 tyramine C00483 tyrosine C01536 UDP C00015 UDP-D-glucose C00029 UDP-N-acetyl-galactosamine/UDP-N-acetyl-glucosamine C00203 / C00043 UMP C00105 uracil C00106 urea C00086 uric acid C00366 uridine C00299 UTP C00075 valine C16436 xanthine C00385 xanthosine C01762 xanthurenic acid C02470 XMP C00655 y-glutamylcysteine C00669 METABOLITES_END #END