#METABOLOMICS WORKBENCH LIUqq140904_20250322_181936 DATATRACK_ID:5768 STUDY_ID:ST003833 ANALYSIS_ID:AN006295 PROJECT_ID:PR002393 VERSION 1 CREATED_ON April 1, 2025, 12:02 pm #PROJECT PR:PROJECT_TITLE Lactate activates trained immunity by fueling the tricarboxylic acid cycle and PR:PROJECT_TITLE regulating histone lactylation PR:PROJECT_SUMMARY Using 13C-based metabolic tracers, we investigate the role of physiologic carbon PR:PROJECT_SUMMARY sources (PCSs) on glucose metabolism in trained immunity. β-glucan treatment PR:PROJECT_SUMMARY led to the rapid conversion of 13C-glucose into lactate and intracellular TCA PR:PROJECT_SUMMARY cycle intermediates (e.g., citrate and fumarate) within the first 8 hours in PR:PROJECT_SUMMARY monocytes. As expected, glucose and glutamine were consumed within 12-24 hrs PR:PROJECT_SUMMARY during the trained immunity process, and PCS treatment mildly affected glucose PR:PROJECT_SUMMARY and glutamine consumption in β-glucan treated monocytes. A slower decrease of PR:PROJECT_SUMMARY other carbon sources, including βOHB, citrate, and pyruvate, was observed in PR:PROJECT_SUMMARY the PCS-supplemented medium compared to the glucose-supplemented medium. PR:INSTITUTE Wuhan University PR:LAST_NAME Liu PR:FIRST_NAME Shi PR:ADDRESS Luojia Hill, Wuchang District, Wuhan, Hubei, 430072, China PR:EMAIL liushi_liushi@whu.edu.cn PR:PHONE 0086-02768754819 #STUDY ST:STUDY_TITLE Lactate activates trained immunity by fueling the tricarboxylic acid cycle and ST:STUDY_TITLE regulating histone lactylation ST:STUDY_SUMMARY Using 13C-based metabolic tracers, we investigate the role of physiologic carbon ST:STUDY_SUMMARY sources (PCSs) on glucose metabolism in trained immunity. β-glucan treatment ST:STUDY_SUMMARY led to the rapid conversion of 13C-glucose into lactate and intracellular TCA ST:STUDY_SUMMARY cycle intermediates (e.g., citrate and fumarate) within the first 8 hours in ST:STUDY_SUMMARY monocytes. As expected, glucose and glutamine were consumed within 12-24 hrs ST:STUDY_SUMMARY during the trained immunity process, and PCS treatment mildly affected glucose ST:STUDY_SUMMARY and glutamine consumption in β-glucan treated monocytes. A slower decrease of ST:STUDY_SUMMARY other carbon sources, including βOHB, citrate, and pyruvate, was observed in ST:STUDY_SUMMARY the PCS-supplemented medium compared to the glucose-supplemented medium. ST:INSTITUTE Wuhan University ST:LAST_NAME Liu ST:FIRST_NAME Shi ST:ADDRESS Luojia Hill, Wuchang District, Wuhan, Hubei, 430072, China ST:EMAIL liushi_liushi@whu.edu.cn ST:PHONE 0086-02768754819 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - Sample1 Sample source:Cultured cells | Treatment:Control RAW_FILE_NAME(Raw file name)=20240402_QC_BEHC18_NEG_MSMS1.raw SUBJECT_SAMPLE_FACTORS - Sample2 Sample source:Cultured cells | Treatment:Control RAW_FILE_NAME(Raw file name)=20240402_QC_BEHC18_NEG_MSMS2.raw SUBJECT_SAMPLE_FACTORS - Sample3 Sample source:Cultured cells | Treatment:Control RAW_FILE_NAME(Raw file name)=20240402_QC_BEHC18_NEG_MSMS3.raw SUBJECT_SAMPLE_FACTORS - QC-1 Sample source:Cultured cells | Treatment:Control RAW_FILE_NAME(Raw file name)=20240402_QC_BEHC18_NEG_MSMS4.raw SUBJECT_SAMPLE_FACTORS - Sample4 Sample source:Cultured cells | Treatment:glucan RAW_FILE_NAME(Raw file name)=20240402_QC_BEHC18_POS_MSMS1.raw SUBJECT_SAMPLE_FACTORS - Sample5 Sample source:Cultured cells | Treatment:glucan RAW_FILE_NAME(Raw file name)=20240402_QC_BEHC18_POS_MSMS2.raw SUBJECT_SAMPLE_FACTORS - Sample6 Sample source:Cultured cells | Treatment:glucan RAW_FILE_NAME(Raw file name)=20240402_QC_BEHC18_POS_MSMS3.raw SUBJECT_SAMPLE_FACTORS - QC-2 Sample source:Cultured cells | Treatment:glucan RAW_FILE_NAME(Raw file name)=20240402_QC_BEHC18_POS_MSMS4.raw SUBJECT_SAMPLE_FACTORS - Sample7 Sample source:Cultured cells | Treatment:glucan_pcs RAW_FILE_NAME(Raw file name)=24NP07020001_BEHC18_NEG.raw SUBJECT_SAMPLE_FACTORS - Sample8 Sample source:Cultured cells | Treatment:glucan_pcs RAW_FILE_NAME(Raw file name)=24NP07020002_BEHC18_NEG.raw SUBJECT_SAMPLE_FACTORS - Sample9 Sample source:Cultured cells | Treatment:glucan_pcs RAW_FILE_NAME(Raw file name)=24NP07020003_BEHC18_NEG.raw SUBJECT_SAMPLE_FACTORS - QC-3 Sample source:Cultured cells | Treatment:glucan_pcs RAW_FILE_NAME(Raw file name)=20240402_QC_BEHC18_NEG_MSMS5.raw #COLLECTION CO:COLLECTION_SUMMARY Monocytes were cultured in modified RPMI medium (MM) containing the indicated CO:COLLECTION_SUMMARY 13C-labeled carbon source and treated with β-glucan (5 μg/ml) for 4 hrs. CO:COLLECTION_PROTOCOL_FILENAME Liu_Shi_Protocol.txt CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Monocytes were cultured in modified RPMI medium (MM) containing the indicated TR:TREATMENT_SUMMARY 13C-labeled carbon source and treated with β-glucan (5 μg/ml) for 4 hrs. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolite extraction: 1. 25 mg tissues for each samples were weighed added into SP:SAMPLEPREP_SUMMARY thickened centrifuge tubes, simultaneously, 2 magnetic beads were added as SP:SAMPLEPREP_SUMMARY well； 2. Add 10 μL of the prepared internal standard 1 to each sample at the SP:SAMPLEPREP_SUMMARY same time; 3. Adding 800 µL of pre-cooled extraction reagent (methanol: SP:SAMPLEPREP_SUMMARY acetonitrile: water (2:2:1, v/v/v)), 50HZ grind for 5min; 4. Place the ground SP:SAMPLEPREP_SUMMARY samples at -20°C for 2h; 5. Centrifuge the sample in a centrifuge（25000 g SP:SAMPLEPREP_SUMMARY *4℃, 15min，Add 600µL of each sample in split-new EP tubes, then freeze dry; SP:SAMPLEPREP_SUMMARY 6. Add 600 μL of 50% methanol to the dried sample, shake until completely SP:SAMPLEPREP_SUMMARY dissolved; 7. Cenrtrifuge at 25000 g *4℃ for 15min，Take the supernatant and SP:SAMPLEPREP_SUMMARY place it in a split-new EP tube; 8. Take 10 μL of each sample and mix into QC SP:SAMPLEPREP_SUMMARY samples. Pass prepared supernatant to LC-MS/MS steps. SP:SAMPLEPREP_PROTOCOL_FILENAME Liu_Shi_protocol.txt #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY In this experiment, waters 2777c UPLC (waters, USA) in series with Q exactive HF CH:CHROMATOGRAPHY_SUMMARY high resolution mass spectrometer (Thermo Fisher Scientific, USA) was used for CH:CHROMATOGRAPHY_SUMMARY the separation and detection of metabolites. Chromatographic separation was CH:CHROMATOGRAPHY_SUMMARY performed on a Waters ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 100 mm, CH:CHROMATOGRAPHY_SUMMARY Waters, USA), and the column temperature was maintained at 45 °C. The mobile CH:CHROMATOGRAPHY_SUMMARY phase consisted of 0.1% formic acid (A) and acetonitrile (B) in the positive CH:CHROMATOGRAPHY_SUMMARY mode, and in the negative mode, the mobile phase consisted of 10 mM ammonium CH:CHROMATOGRAPHY_SUMMARY formate (A) and acetonitrile (B). The gradient conditions were as follows: 0-1 CH:CHROMATOGRAPHY_SUMMARY min, 2% B; 1-9 min, 2%-98% B; 9- 12 min, 98% B; 12-12.1 min, 98% B to 2% B; and CH:CHROMATOGRAPHY_SUMMARY 12.1-15min, 2% B. The flow rate was 0.35 mL/min and the injection volume was 5 CH:CHROMATOGRAPHY_SUMMARY μL. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 0-1 min, 2% B; 1-9 min, 2%-98% B; 9- 12 min, 98% B; 12-12.1 min, 98% B to 2% B; CH:FLOW_GRADIENT and 12.1-15min, 2% B CH:FLOW_RATE 0.35 mL/min CH:COLUMN_TEMPERATURE 45 CH:METHODS_FILENAME Liu_Shi_Protocol.txt #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Liu_Shi_Protocol.txt #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Mass spectrometry conditions:Using Q Exactive HF(Thermo Fisher Scientific, USA) MS:MS_COMMENTS perform primary and secondary mass spectrometry data acquisition. The full scan MS:MS_COMMENTS range was 70‒1050 m/z with a resolution of 120,000, and the automatic gain MS:MS_COMMENTS control (AGC) target for MS acquisitions was set to 3e6 with a maximum ion MS:MS_COMMENTS injection time of 100 ms. Top 3 precursors were selected for subsequent MSMS MS:MS_COMMENTS fragmentation with a maximum ion injection time of 50 ms and resolution of MS:MS_COMMENTS 30000, the AGC was 1e5. The stepped normalized collision energy was set to 20, MS:MS_COMMENTS 40 and 60 eV. ESI parameters were setting as : Sheath gas flow rate was 40, Aux MS:MS_COMMENTS gas flow rate was 10， positive-ion mode Spray voltage(|KV|) was 3.80, MS:MS_COMMENTS negative-ion mode Spray voltage(|KV|) was 3.20， Capillary temperature was MS:MS_COMMENTS 320°C， Aux gas heater temperature was 350°C. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak intensity MS_METABOLITE_DATA_START Samples Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Factors Sample source:Cultured cells | Treatment:Control Sample source:Cultured cells | Treatment:Control Sample source:Cultured cells | Treatment:Control Sample source:Cultured cells | Treatment:glucan Sample source:Cultured cells | Treatment:glucan Sample source:Cultured cells | Treatment:glucan Sample source:Cultured cells | Treatment:glucan_pcs Sample source:Cultured cells | Treatment:glucan_pcs Sample source:Cultured cells | Treatment:glucan_pcs Inosinic acid 72919.7051618148 93027.4418599774 175519.21833995 22744.1873969167 34407.3764673562 30551.2738831448 5.09149891172973E7 1.66899483693468E7 2.44239226232204E7 Citric acid 1032970.68066103 9793118.59838022 1145919.02349609 1.12823473455141E7 1124982.72829331 2374320.15439096 1.44918029822102E7 2818540.90128405 4594851.93438099 Cytidine 3011851.55511574 2003412.24087066 3105939.21595873 2071410.55729622 2520153.22981965 2877435.18122839 1152427.31516009 1366103.33446164 1255790.50263583 L-Serine 5387619.77819671 5904237.65151171 4608269.84721537 7523827.97452602 6175411.86753667 5812715.71070046 5073035.87522759 4907153.55387237 5801753.51490209 Adenine 1.31801262142635E7 1.15022322090614E7 1.34432121682839E7 1320128.0050335 9894974.65872648 7738087.18192352 4744035.34094256 700850.358833616 5538552.17446359 L-Tryptophan 8593032.62713725 1.06569821726386E7 7357567.10918329 1.55551629354794E7 9106986.02639936 9206061.55260261 9329246.87962318 7871328.71203953 1.04517404584693E7 Uridine 8734309.81761871 1.44371439544649E7 1.15059581789074E7 1.57551523241308E7 5653635.87718278 6513436.53587078 9216222.21492979 5419668.1374868 1.50154794635573E7 Succinic acid 1834166.30332351 2007690.57738149 1353074.90766901 1483917.44421837 5768534.97522871 4849252.62955497 536168.995902027 3800525.25610032 467871.594834194 L-Aspartic acid 3.38633195248475E7 3.75529765342883E7 7.49380469819717E7 5.05071955492441E7 5.95657981435756E7 4.00233193478537E7 7.86630938579099E7 8.81894026374087E7 8.76162629600638E7 Hypoxanthine 9.22142715509079E7 1.07949176352607E8 9.73046340188087E7 7.49862834376647E7 8.01468840844917E7 7.28814816285118E7 5.09549664109066E7 1.1167612970616E8 8.33794864091196E7 L-Tyrosine 8404982.1089503 1.04627427906556E7 9113674.43308335 1.49138747097222E7 1.14903988489031E7 1.17861205716789E7 7688950.76461235 1.12593830426231E7 9488645.03264334 Fumaric acid 2.67669773558266E7 2.78377822251967E7 2.12797031558689E7 2.94457463913671E7 2.77649218493114E7 2.30646538386192E7 1.9823753972071E7 1.37617305403968E7 1.86592426470041E7 Pantothenic acid 3.77406549640987E7 3.81336861437085E7 3.03899824409374E7 4.30427369321652E7 3.92545319547678E7 3.3199330644923E7 3.11170663874311E7 5.98486644355871E7 3.16554706600167E7 Methylglutaric acid 2474644.28744815 7473929.94867868 4.27032384377682E7 7830621.555 8280144.75229612 9098063.0673801 1.45189495588552E7 1.07310829774011E7 2.35916571434487E7 Palmitoleic acid 1.40649208451514E7 1.23985389840744E7 1.05702611633302E7 9664372.50572883 8974341.81057959 9621258.14475872 7384572.27130131 1.15444411009208E7 1.98034592970949E7 Myristic acid 1493715.54859641 1322907.64474348 1308723.15585023 1486477.29496678 1211752.52072032 1261841.39845344 1270377.63055756 1427012.22261426 2667196.52823329 3-Hydroxybutyric acid 5948166.08672553 5692330.37898362 7778515.88615851 7909108.77829348 7909108.77829348 4960401.49385832 9170590.83072364 9597087.8249709 8048174.80097075 L-Proline 2117212.31205643 2833127.906 1947371.43477459 4173276.25538787 2521397.60801314 2694869.16616669 1678965.14383566 1983375.70516277 2178837.96097603 N-Acetyl-L-methionine 1577536.49310738 1888992.1064568 1243839.27956939 1702243.74852284 3259144.39406427 1922546.65263253 2821432.49041862 4752254.64446421 2425286.77985232 L-Lactic acid 3.64084712632599E8 4.31813077283997E8 2.06775314796478E8 6.71880739857218E8 4.40285701540983E8 5.0488707180118E8 3.61087880427511E8 4.59379331942247E8 3.43824160053079E8 p-Cresol sulfate 3764135.16165571 3626458.75596069 6286602.24146835 9201090.63251677 4090761.4042098 1.54361175492611E7 2245126.99609468 3964117.69065659 2694667.39742033 4-Vinylphenol sulfate 1238833.50986668 2682098.40042972 1441760.54976997 1994354.49017289 195019.801191556 441872.231333465 900184.173594825 393588.644740057 1214844.99024817 3-Hydroxyvalproic acid 414031.308182792 329800.101812589 509835.743099448 539325.900212874 832983.302101234 379230.829596972 1835277.66208771 1067863.98247308 1180935.85555084 Taurochenodeoxycholic acid 12852.5778662879 23894.1810846871 12696.6472879279 253010.545142996 4076.45525529138 19492.4413673398 1859410.12301436 23331.242035151 937790.132271327 Tauro-b-muricholic acid 40199.546334079 126533.817833999 131726.31324405 1054012.84884089 26362.8763932998 37091.9395811803 3.00428191288994E7 55143.2704803521 1.91598273401881E7 Linoleic acid 3.49196561980888E8 4.0287947283391E8 3.93282769720983E8 2.32319940808032E8 3.65360063756604E8 2.50698389703805E8 3.06469389339866E8 2.79246159084356E8 3.03180662108907E8 2-hydroxylaurate 1110856.94338653 647309.737854885 1269170.44981981 729381.509123016 1721126.84488265 528254.328442729 8960948.50768398 6031570.77856815 3329790.27320958 xi-10-Hydroxyoctadecanoic acid 2347625.98560009 1841877.19299196 2842846.98983116 1579025.2923775 3049848.46294267 1138853.41126771 3060813.50372248 2060051.72766254 2709126.54524634 N-Palmitoyltaurine 1119760.59924021 903555.627578451 1351786.10904307 905177.423843379 1770874.68329873 1339101.33630566 1674182.60575091 2273442.73009555 1674358.79070868 PE(P-18:1/0:0) 5530008.54933115 6857931.9739384 3648795.14089989 409544.252103637 1907285.04197385 3199512.91799081 7512090.01574572 8226508.30827594 5502970.16808735 PG(18:1/0:0) 1.05485038774395E7 5808348.62090215 7598273.49886603 4418547.11912739 8122758.37198517 6396886.0995201 5337420.91617765 7109935.55475479 4108123.89977787 PG(18:0/0:0) 6177394.3302473 2583667.74292087 4243032.56255689 1035689.64160819 8590193.45156388 3182453.58460697 2777373.22745192 5908930.03510488 2311348.22778648 PI(18:2/0:0) 4436569.16361846 3684234.73775051 6425253.55292095 2148633.04079224 4386531.84307473 3302664.88236231 1860192.6759753 3196579.1106322 1622472.43909487 LysoPI(18:0/0:0) 1.10820471023864E7 1.20646480663262E7 1.08479941262552E7 5201909.53654979 1.17913134753833E7 8135767.92881507 5036195.82770782 1.0440527220553E7 8852465.05319495 PG(16:0/0:0) 2.29565514706953E7 1.28884222824624E7 1.60299259948664E7 9367021.10034837 2.93134492325753E7 2.40161743581993E7 1.51007817497268E7 2.83279957627556E7 1.47121412071357E7 5-Aminovaleric acid 1.13858962585805E7 1.19455088807005E7 1.05659243927643E7 1.82151529515376E7 1.31738598208732E7 1.64614086125394E7 9883867.69787295 1.04688960956436E7 1.08525128816474E7 4-Oxoproline 2.04217488076348E7 2.71881765557361E7 3.31270958910569E7 1.68585217274368E7 9124793.43010099 1.77244056631568E7 2.24063937513807E7 2.50886934272688E7 2.08819176535982E7 3-Hydroxydecanoic acid 975447.745046284 671324.838244352 1241800.69909842 1031473.77957133 2058727.91205004 651793.98281095 9141774.80038066 5464401.23258445 4021639.71516289 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z HMDB ID Inosinic acid 0.7 347.03971 HMDB0000175 Citric acid 0.569 191.01972 HMDB0000094 Cytidine 1.042 242.07806 HMDB0000089 L-Serine 0.638 104.03533 HMDB0000187 Adenine 1.684 134.04731 HMDB0000034 L-Tryptophan 3.098 203.08262 HMDB0000929 Uridine 1.238 243.06196 HMDB0000296 Succinic acid 0.622 117.0194 HMDB0000254 L-Aspartic acid 0.621 132.03031 HMDB0000191 Hypoxanthine 1.074 135.03134 HMDB0000157 L-Tyrosine 1.056 180.06663 HMDB0000158 Fumaric acid 0.619 115.00373 HMDB0000134 Pantothenic acid 1.234 218.10324 HMDB0000210 Methylglutaric acid 0.706 145.05074 HMDB0000752 Palmitoleic acid 9.912 253.21712 HMDB0003229 Myristic acid 9.79 227.20161 HMDB0000806 3-Hydroxybutyric acid 0.698 103.04006 HMDB0000011 L-Proline 0.698 114.05604 HMDB0000162 N-Acetyl-L-methionine 2.372 190.05436 HMDB0011745 L-Lactic acid 0.654 89.02441 HMDB0000190 p-Cresol sulfate 4.158 187.00707 HMDB0011635 4-Vinylphenol sulfate 4.785 199.00703 HMDB0062775 3-Hydroxyvalproic acid 5.15 159.10275 HMDB0013899 Taurochenodeoxycholic acid 8.528 498.28909 HMDB0000951 Tauro-b-muricholic acid 7.944 514.28357 HMDB0000932 Linoleic acid 10.065 279.23266 HMDB0000673 2-hydroxylaurate 8.218 215.16513 xi-10-Hydroxyoctadecanoic acid 10.05 299.25909 HMDB0037396 N-Palmitoyltaurine 9.691 362.23719 HMDB0240594 PE(P-18:1/0:0) 10.25 462.29879 PG(18:1/0:0) 9.838 509.28808 PG(18:0/0:0) 10.144 511.30361 PI(18:2/0:0) 9.548 595.28932 LysoPI(18:0/0:0) 10.107 599.32054 HMDB0240261 PG(16:0/0:0) 9.758 483.27261 5-Aminovaleric acid 0.715 116.07171 HMDB0003355 4-Oxoproline 0.674 128.03535 HMDB0304793 3-Hydroxydecanoic acid 7.062 187.13398 HMDB0094656 METABOLITES_END #END