#METABOLOMICS WORKBENCH lgafeira_20240508_075633 DATATRACK_ID:4821 STUDY_ID:ST003202 ANALYSIS_ID:AN005252 PROJECT_ID:PR001996 VERSION 1 CREATED_ON May 14, 2024, 9:03 am #PROJECT PR:PROJECT_TITLE Integrated multi-omics unveil the impact of the phosphinic compounds, PR:PROJECT_TITLE desmethylphosphinothricin and its keto-analogue, on Escherichia coli metabolism PR:PROJECT_SUMMARY Desmethylphosphinothricin (L-Glu-γ-PH) is the phosphinic analogue of glutamate PR:PROJECT_SUMMARY with a carbon-phosphorus (C-P) bond. In L-Glu-γ-PH the phosphinic group acts as PR:PROJECT_SUMMARY a bioisostere of glutamate γ-carboxyl group allowing the molecule to be a PR:PROJECT_SUMMARY substrate of Escherichia coli glutamate decarboxylase, a pyridoxal PR:PROJECT_SUMMARY 5’-phosphate (PLP)-dependent α-decarboxylase. In addition, the L-Glu-γ-PH PR:PROJECT_SUMMARY decarboxylation product, GABA-PH, is further metabolized by bacterial PR:PROJECT_SUMMARY GABA-transaminase, another PLP-dependent enzyme, and succinic semialdehyde PR:PROJECT_SUMMARY dehydrogenease, a NADP+ -dependent enzyme. The product of these consecutive PR:PROJECT_SUMMARY reactions, the so-called GABA shunt, lead to the formation of succinate-PH, the PR:PROJECT_SUMMARY phosphinic analogue of succinate, a TCA cycle intermediate. Notably, L-Glu-γ-PH PR:PROJECT_SUMMARY displays an antibacterial activity of the same order of well-established PR:PROJECT_SUMMARY antibiotics in E. coli. The dipeptide L-Leu-Glu-γ-PH was shown to display a PR:PROJECT_SUMMARY higher efficacy, likely as a consequence of an improved penetration into the PR:PROJECT_SUMMARY bacteria. Herein, with the aim of further understanding the intracellular PR:PROJECT_SUMMARY effects of L-Glu-γ-PH, 1H NMR-based metabolomics and LC-MS-based shotgun PR:PROJECT_SUMMARY proteomics were used. This study included also the keto-analogue of L-Glu-γ-PH, PR:PROJECT_SUMMARY α-ketoglutarate-γ-PH (α-KG-γ-PH), which also exhibits antimicrobial PR:PROJECT_SUMMARY activity. L-Glu-γ-PH and α-KG-γ-PH were found to similarly impact the PR:PROJECT_SUMMARY bacterial metabolism, though the overall effect of α-KG-γ-PH is more PR:PROJECT_SUMMARY pervasive, and not exclusively because of its intracellular conversion into PR:PROJECT_SUMMARY L-Glu-γ-PH. Notably, both molecules impact the pathways where aspartate, PR:PROJECT_SUMMARY glutamate and glutamine are used as precursors for the biosynthesis of related PR:PROJECT_SUMMARY metabolites, activate the acid stress response and deprive cells of nitrogen. PR:PROJECT_SUMMARY This work highlights the multi-target drug potential of L-Glu-γ-PH and PR:PROJECT_SUMMARY α-KG-γ-PH and paves the way for their exploitation as antimicrobials. PR:INSTITUTE ITQB NOVA PR:LAST_NAME Gonçalves PR:FIRST_NAME Luís G PR:ADDRESS Avenida Republica, Oeiras, Not USCanada, 2780-157 Oeiras, Portugal PR:EMAIL lgafeira@itqb.unl.pt PR:PHONE 214469464 #STUDY ST:STUDY_TITLE Integrated multi-omics unveil the impact of the phosphinic compounds, ST:STUDY_TITLE desmethylphosphinothricin and its keto-analogue, on Escherichia coli metabolism ST:STUDY_SUMMARY Desmethylphosphinothricin (L-Glu-γ-PH) is the phosphinic analogue of glutamate ST:STUDY_SUMMARY with a carbon-phosphorus (C-P) bond. In L-Glu-γ-PH the phosphinic group acts as ST:STUDY_SUMMARY a bioisostere of glutamate γ-carboxyl group allowing the molecule to be a ST:STUDY_SUMMARY substrate of Escherichia coli glutamate decarboxylase, a pyridoxal ST:STUDY_SUMMARY 5’-phosphate (PLP)-dependent α-decarboxylase. In addition, the L-Glu-γ-PH ST:STUDY_SUMMARY decarboxylation product, GABA-PH, is further metabolized by bacterial ST:STUDY_SUMMARY GABA-transaminase, another PLP-dependent enzyme, and succinic semialdehyde ST:STUDY_SUMMARY dehydrogenease, a NADP+ -dependent enzyme. The product of these consecutive ST:STUDY_SUMMARY reactions, the so-called GABA shunt, lead to the formation of succinate-PH, the ST:STUDY_SUMMARY phosphinic analogue of succinate, a TCA cycle intermediate. Notably, L-Glu-γ-PH ST:STUDY_SUMMARY displays an antibacterial activity of the same order of well-established ST:STUDY_SUMMARY antibiotics in E. coli. The dipeptide L-Leu-Glu-γ-PH was shown to display a ST:STUDY_SUMMARY higher efficacy, likely as a consequence of an improved penetration into the ST:STUDY_SUMMARY bacteria. Herein, with the aim of further understanding the intracellular ST:STUDY_SUMMARY effects of L-Glu-γ-PH, 1H NMR-based metabolomics and LC-MS-based shotgun ST:STUDY_SUMMARY proteomics were used. This study included also the keto-analogue of L-Glu-γ-PH, ST:STUDY_SUMMARY α-ketoglutarate-γ-PH (α-KG-γ-PH), which also exhibits antimicrobial ST:STUDY_SUMMARY activity. L-Glu-γ-PH and α-KG-γ-PH were found to similarly impact the ST:STUDY_SUMMARY bacterial metabolism, though the overall effect of α-KG-γ-PH is more ST:STUDY_SUMMARY pervasive, and not exclusively because of its intracellular conversion into ST:STUDY_SUMMARY L-Glu-γ-PH. Notably, both molecules impact the pathways where aspartate, ST:STUDY_SUMMARY glutamate and glutamine are used as precursors for the biosynthesis of related ST:STUDY_SUMMARY metabolites, activate the acid stress response and deprive cells of nitrogen. ST:STUDY_SUMMARY This work highlights the multi-target drug potential of L-Glu-γ-PH and ST:STUDY_SUMMARY α-KG-γ-PH and paves the way for their exploitation as antimicrobials. ST:INSTITUTE ITQB NOVA ST:LAST_NAME Gonçalves ST:FIRST_NAME Luís ST:ADDRESS Avenida Republica, Oeiras, Not USCanada, 2780-157 Oeiras, Portugal ST:EMAIL lgafeira@itqb.unl.pt ST:PHONE 214469464 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Escherichia coli SU:TAXONOMY_ID 562 #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 - A Sample source:E. coli | Condition:Control RAW_FILE_NAME(raw data)=A SUBJECT_SAMPLE_FACTORS - B Sample source:E. coli | Condition:Control RAW_FILE_NAME(raw data)=B SUBJECT_SAMPLE_FACTORS - AE Sample source:E. coli | Condition:Control RAW_FILE_NAME(raw data)=AE SUBJECT_SAMPLE_FACTORS - AF Sample source:E. coli | Condition:Control RAW_FILE_NAME(raw data)=AF SUBJECT_SAMPLE_FACTORS - AM Sample source:E. coli | Condition:Control RAW_FILE_NAME(raw data)=AM SUBJECT_SAMPLE_FACTORS - AN Sample source:E. coli | Condition:Control RAW_FILE_NAME(raw data)=AN SUBJECT_SAMPLE_FACTORS - AO Sample source:E. coli | Condition:Control RAW_FILE_NAME(raw data)=AO SUBJECT_SAMPLE_FACTORS - AP Sample source:E. coli | Condition:Control RAW_FILE_NAME(raw data)=AP SUBJECT_SAMPLE_FACTORS - G Sample source:E. coli | Condition:Control RAW_FILE_NAME(raw data)=G SUBJECT_SAMPLE_FACTORS - H Sample source:E. coli | Condition:Control RAW_FILE_NAME(raw data)=H SUBJECT_SAMPLE_FACTORS - AA Sample source:E. coli | Condition:L-Glutamate-y-PH RAW_FILE_NAME(raw data)=AA SUBJECT_SAMPLE_FACTORS - AG Sample source:E. coli | Condition:L-Glutamate-y-PH RAW_FILE_NAME(raw data)=AG SUBJECT_SAMPLE_FACTORS - AH Sample source:E. coli | Condition:L-Glutamate-y-PH RAW_FILE_NAME(raw data)=AH SUBJECT_SAMPLE_FACTORS - C Sample source:E. coli | Condition:L-Glutamate-y-PH RAW_FILE_NAME(raw data)=C SUBJECT_SAMPLE_FACTORS - D Sample source:E. coli | Condition:L-Glutamate-y-PH RAW_FILE_NAME(raw data)=D SUBJECT_SAMPLE_FACTORS - I Sample source:E. coli | Condition:L-Glutamate-y-PH RAW_FILE_NAME(raw data)=I SUBJECT_SAMPLE_FACTORS - Q Sample source:E. coli | Condition:L-Glutamate-y-PH RAW_FILE_NAME(raw data)=Q SUBJECT_SAMPLE_FACTORS - R Sample source:E. coli | Condition:L-Glutamate-y-PH RAW_FILE_NAME(raw data)=R SUBJECT_SAMPLE_FACTORS - Z Sample source:E. coli | Condition:L-Glutamate-y-PH RAW_FILE_NAME(raw data)=Z SUBJECT_SAMPLE_FACTORS - AB Sample source:E. coli | Condition:a-Ketoglutarate-PH RAW_FILE_NAME(raw data)=AB SUBJECT_SAMPLE_FACTORS - AC Sample source:E. coli | Condition:a-Ketoglutarate-PH RAW_FILE_NAME(raw data)=AC SUBJECT_SAMPLE_FACTORS - AI Sample source:E. coli | Condition:a-Ketoglutarate-PH RAW_FILE_NAME(raw data)=AI SUBJECT_SAMPLE_FACTORS - AL Sample source:E. coli | Condition:a-Ketoglutarate-PH RAW_FILE_NAME(raw data)=AL SUBJECT_SAMPLE_FACTORS - E Sample source:E. coli | Condition:a-Ketoglutarate-PH RAW_FILE_NAME(raw data)=E SUBJECT_SAMPLE_FACTORS - F Sample source:E. coli | Condition:a-Ketoglutarate-PH RAW_FILE_NAME(raw data)=F SUBJECT_SAMPLE_FACTORS - M Sample source:E. coli | Condition:a-Ketoglutarate-PH RAW_FILE_NAME(raw data)=M SUBJECT_SAMPLE_FACTORS - N Sample source:E. coli | Condition:a-Ketoglutarate-PH RAW_FILE_NAME(raw data)=N SUBJECT_SAMPLE_FACTORS - S Sample source:E. coli | Condition:a-Ketoglutarate-PH RAW_FILE_NAME(raw data)=S SUBJECT_SAMPLE_FACTORS - T Sample source:E. coli | Condition:a-Ketoglutarate-PH RAW_FILE_NAME(raw data)=T #COLLECTION CO:COLLECTION_SUMMARY E. coli K12 strain MG1655 cultures, were started from from overnight CO:COLLECTION_SUMMARY pre-cultures. Three growths conditions were performed: control, in the presence CO:COLLECTION_SUMMARY of L-Glu-γ-PH and in the presence of α-KG-γ-PH. Phosphinic compounds CO:COLLECTION_SUMMARY concentrations chosen were close to MIC60, i.e. 3.5 µg/ml and 8.5 µg/ml, CO:COLLECTION_SUMMARY respectively. Bacterial cultures were incubated at 37 °C, 150 rpm until CO:COLLECTION_SUMMARY late-exponential phase (OD600 = 1.0), which was reached after 17.4 ± 0.9, 24.0 CO:COLLECTION_SUMMARY ± 2.5 and 26.0 ± 3.0 hours for the untreated cultures, the L-Glu-γ-PH-treated CO:COLLECTION_SUMMARY and the α-KG-γ-PH-treated cultures, respectively. CO:COLLECTION_PROTOCOL_FILENAME LG_protocol.txt CO:SAMPLE_TYPE E. coli #TREATMENT TR:TREATMENT_SUMMARY Samples were split in two 50-ml falcon tubes and immediately transferred to a TR:TREATMENT_SUMMARY -35 °C bath (Julabo FP50‐HE) for 2.5 minutes, for metabolism quenching. Then, TR:TREATMENT_SUMMARY the tubes were centrifuged for 5 min at 5,000 x g at 4 °C, to obtain the TR:TREATMENT_SUMMARY bacterial pellets to which two different protocols were applied in order to TR:TREATMENT_SUMMARY obtain samples for metabolomic and proteomic analysis. The cell pellets were TR:TREATMENT_SUMMARY washed in 1.5 ml of cold physiological solution, then transferred in a 2-ml TR:TREATMENT_SUMMARY eppendorf tube and centrifuged for 5 min at 5,000 x g at 4 °C; the supernatants TR:TREATMENT_SUMMARY were discarded, and each pellet was resuspended in 1 ml of cold physiological TR:TREATMENT_SUMMARY solution. The OD600 was measured using a 5-µl aliquot diluted 1:200. Based on TR:TREATMENT_SUMMARY the OD600 readings, the volume corresponding to OD600 = 25 (~350 µl) was TR:TREATMENT_SUMMARY transferred to another 2 ml eppendorf tube and centrifuged for 5 min at 5,000 x TR:TREATMENT_SUMMARY g, at 4 °C. The physiological solution was discarded, and the pellets TR:TREATMENT_SUMMARY immediately frozen in liquid nitrogen and stored at -20 °C. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Pellets stored for metabolomics studies were thawed on ice, resuspended in 750 SP:SAMPLEPREP_SUMMARY µl of ice-cold methanol (60% v/v), submitted to three freeze‐thaw cycles SP:SAMPLEPREP_SUMMARY using liquid nitrogen, and centrifuged for 5 minutes at 12,900 x g, at 4 °C. SP:SAMPLEPREP_SUMMARY The supernatants were transferred into clean 1.5-ml eppendorf tubes, and the SP:SAMPLEPREP_SUMMARY extraction procedure was repeated a second time on the residual pellet. Samples SP:SAMPLEPREP_SUMMARY were then concentrated down to dryness using a speed‐vacuum concentrator SP:SAMPLEPREP_SUMMARY (Vacufuge® plus, Eppendorf) and stored at -20 °C until use. Dried samples were SP:SAMPLEPREP_SUMMARY dissolved in 250 µl of potassium phosphate buffer 0.35 M, pH 7.0, 451.5 µl D2O SP:SAMPLEPREP_SUMMARY with 2 mM sodium azide and 50 µl D2O with 3.2 mM TSP (final volume 751.5 µl SP:SAMPLEPREP_SUMMARY per sample). Subsequently, 600 µl of each sample was transferred to a 5 mm NMR SP:SAMPLEPREP_SUMMARY tube for 1H NMR spectra acquisition. For 31P NMR, after 1H NMR acquisition, SP:SAMPLEPREP_SUMMARY samples were pooled, concentrated by speed-vacuum concentrator (as above) and SP:SAMPLEPREP_SUMMARY dissolved in 1 ml of D2O and transferred to a new 5 mm tube. Prior to 31P NMR SP:SAMPLEPREP_SUMMARY spectra acquisition, samples were measured for pH, which ranged between 7.1-7.4. #ANALYSIS AN:ANALYSIS_PROTOCOL_FILE LG_protocol.txt #NMR NM:INSTRUMENT_NAME Bruker Avance II+ 800 MHz NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 800 #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS nmol NMR_METABOLITE_DATA_START Samples A B AE AF AM AN AO AP G H AA AG AH C D I Q R Z AB AC AI AL E F M N S T Factors Sample source:E. coli | Condition:Control Sample source:E. coli | Condition:Control Sample source:E. coli | Condition:Control Sample source:E. coli | Condition:Control Sample source:E. coli | Condition:Control Sample source:E. coli | Condition:Control Sample source:E. coli | Condition:Control Sample source:E. coli | Condition:Control Sample source:E. coli | Condition:Control Sample source:E. coli | Condition:Control Sample source:E. coli | Condition:L-Glutamate-y-PH Sample source:E. coli | Condition:L-Glutamate-y-PH Sample source:E. coli | Condition:L-Glutamate-y-PH Sample source:E. coli | Condition:L-Glutamate-y-PH Sample source:E. coli | Condition:L-Glutamate-y-PH Sample source:E. coli | Condition:L-Glutamate-y-PH Sample source:E. coli | Condition:L-Glutamate-y-PH Sample source:E. coli | Condition:L-Glutamate-y-PH Sample source:E. coli | Condition:L-Glutamate-y-PH Sample source:E. coli | Condition:a-Ketoglutarate-PH Sample source:E. coli | Condition:a-Ketoglutarate-PH Sample source:E. coli | Condition:a-Ketoglutarate-PH Sample source:E. coli | Condition:a-Ketoglutarate-PH Sample source:E. coli | Condition:a-Ketoglutarate-PH Sample source:E. coli | Condition:a-Ketoglutarate-PH Sample source:E. coli | Condition:a-Ketoglutarate-PH Sample source:E. coli | Condition:a-Ketoglutarate-PH Sample source:E. coli | Condition:a-Ketoglutarate-PH Sample source:E. coli | Condition:a-Ketoglutarate-PH 2-Heptanone 27.1 26.6 31.5 32 31.8 28 34 35.1 27.7 27.9 34.7 29.9 22.1 22.5 29.1 27.3 27.7 28.4 22.8 30.2 42.7 23.4 21.8 28.2 25.3 26.5 31 22.8 22.6 3-Hydroxy-3-methylglutarate 3.1 3.9 6.7 4.9 2.6 5.2 4.1 4.2 5.4 3.9 3.4 3.2 2.8 3 4.2 3 3.4 3.1 2.9 4.1 4 4 5.1 4 4.8 4.6 4 4.1 4.3 4-Aminobutyrate 127 76.9 17.2 12.7 13.2 13.3 14.4 17.9 83.1 55.2 109.3 109 90.7 132.9 334.6 134.2 171.3 135.8 120.7 235.3 177.2 141.4 144.6 210.9 208.3 206.4 206.9 310.6 415.6 Acetate 152.6 143.1 67.3 77.3 50.2 69.3 85.5 88.1 159.2 107.2 104.7 85.7 104.8 96.8 95 109.2 120.2 132.1 81 130.7 128.6 106.7 95.7 115.3 107 145.1 114.9 91.3 146.6 Acetoin 5.2 4.1 4.4 3.3 2.9 4 4 4.5 5 3.9 3.7 3.8 3.5 3.2 3.3 3.3 4.7 4.1 3.4 4.8 4.5 4.6 4.3 4.3 4.5 5 5.5 4.1 4.1 Acetone 4.5 5.3 7.3 3.8 4.3 4 8.6 7.2 4.9 13.5 7.8 3.9 4.3 4.5 5.7 6.3 5.6 4.9 4.6 3.8 5 4.2 3.7 4.4 7.9 4.7 5.4 4.6 4 ADP 15 16.3 8.8 10.5 10.3 8.7 12.5 10.5 22.6 13.4 16.1 17.2 19.2 14.3 11.8 22.1 14.3 14.7 16.4 7.8 10.5 9.6 11.9 7.4 6.8 10.8 7.9 7.8 7.7 Alanine 51 46.2 43.1 36 79.3 54.9 50.9 62 57.3 36 83.1 47 28.9 40.5 163.8 47.4 97.1 95.2 78.7 82.8 98.1 68.5 44.9 52 61.9 53.3 50.6 75.5 93.2 AMP 9.3 9.6 0.7 1.7 3.2 1.6 4.1 1.6 7.7 8 5.8 5.9 1.8 11.9 16.3 12.1 15.2 16.2 8.4 19.7 16.9 23.3 16.8 22.7 16.9 20.5 20.4 17.2 19.2 Arginine 3.5 3.6 2.6 1.6 2.7 2.4 2.5 1.5 2.2 2.2 2.6 3.7 2.2 1.8 1.5 1.9 2.3 1.7 2.9 5.9 1.6 3.5 1.5 2.3 1.5 2.7 1.9 1.4 1.4 Aspartate 3.5 4.5 2.5 5.6 22.8 4.4 9 4.8 9.2 20.8 15.3 17.9 12.2 17.1 26.9 11.8 14.9 23.8 15.1 ATP 3.2 3.4 2.5 2.3 2.5 2 3.4 3.3 3.4 3.2 3.8 3.4 3.9 4.5 4 5.6 4.6 5.3 3.5 2.6 2.2 2.6 2 2.3 2.7 2.4 2.3 2 1.7 Choline 2.5 2.6 1.3 1.2 2.5 1.8 1.7 2 2.4 1.1 1.1 0.9 0.4 0.7 1.8 0.8 1.2 1 1.2 1.8 1.3 1.7 1 1.4 1.5 1.2 1.2 1.5 1.6 Citrate 42.7 40.7 32.4 32 26 29.9 31.6 30.7 39.4 32.7 52.3 58.8 48.1 62.4 58 58.1 53.3 55.5 50.1 33.3 34.3 30.6 26.5 34.2 25.9 38.3 34 29 28.2 Coenzyme A 131.9 123.8 88.2 98 86.7 77.6 92.1 79.9 112.9 107 118.2 103.1 81.9 121 144.1 108.3 130.2 141.1 123.5 109.6 97.4 85.1 74.3 104 77.7 113.4 116.9 90.9 92.3 Cytidine 4.7 5.6 4.4 3.6 6.2 4.7 2.1 1.2 2.3 5.8 2.2 2.1 5.2 6.1 Cytidine monophosphate 12 11.7 2.8 6.3 4.7 7.6 3.5 5.6 3.4 14.8 9.2 7.6 11.6 5.7 9.9 10.9 12.4 9 13.5 7.8 13.1 10.2 6.8 7.9 dCMP 25.6 18 8.7 24.3 32.3 22.1 32.4 56.9 42.6 52.7 40.1 48 57.3 38.8 60.1 37.8 35.4 Ethanol 18.9 16.4 17.2 15.3 16.9 16.7 16.9 17.8 18.9 21.4 14.8 13.8 17.6 19.6 14.2 19.8 17.8 13.7 15.3 14.8 15.4 12.6 14.3 15.3 17 14.2 17.4 16.2 13.3 Formate 3.5 3.9 2.8 2.2 6.1 6.4 4 17.6 2.9 3.2 2.8 3.5 2.3 2.8 3.9 3.1 8.6 3 2.8 3.9 2.9 8.7 2.4 3.2 5.4 5.2 2.6 3.1 15.1 Fumarate 6.4 4.8 7.9 7.4 11.9 10.7 10.7 7.9 9.2 5.1 2.9 3.6 1.9 2 2.6 2.6 2.9 2.7 2.6 5.1 3.9 3.1 2.1 1.6 1.5 2 1.3 3.1 4.1 Glucose 10.3 8.8 6.1 6.9 4.6 6.3 5.8 10.6 8.5 8.7 3.7 6 4.7 4.9 9 7.3 9.4 8.5 5.5 43.7 12 23.2 13 12.9 32.2 14.9 15.2 32.6 39.4 Glucose-1-phosphate 13.4 13.4 10.4 8.9 9.1 8 9.5 8.1 16.1 11.6 6.7 6.3 7.2 8.3 7.2 8.2 7.9 8.2 5.5 7 11.4 5.6 7.4 7.4 5.7 8.5 7.2 4 8.9 Glucose-6-phosphate 26.8 33.9 19.8 23.4 14.8 12.8 23 15.5 39.7 31.3 16.3 16.4 12.6 14.7 17.2 25.1 20.6 16.8 17.8 65 62.1 58.2 48.8 52.3 114.1 54.9 53.3 43.8 46.8 Glutamate 69.6 58.2 65.5 73.8 124.3 104.8 105.9 107.4 74 49.6 194.1 122 34.5 34 129.7 34.1 196.3 268.1 192.8 355.6 297.3 319 101.4 59.1 123 63.4 58.3 152.4 76.7 Glutamine 42.6 60.7 42.2 45.8 57.1 50.3 44.7 40.8 57.8 39.6 29.1 82.5 41.5 67.3 65.8 66.2 89.1 92.3 99.3 34.2 29.9 76.7 71 54.7 67.3 60.4 64.6 46.6 49.8 Glutathione 48 50.4 19.1 19 69.1 24.7 25.2 21.3 22.8 16.9 195.8 128.8 41.2 61.2 206.9 61.3 202.9 225.7 202.6 170.1 154.9 188.9 145.5 141.6 137.1 121.7 136.6 152 163.4 Glycine 25.3 23.9 19.2 20.9 23.1 22.4 25.8 23.4 24.6 21.5 31.8 31.1 26.6 38.6 35.1 33.1 32.6 32.2 23 31.5 25.6 31.4 27.5 26.1 28 28.2 32.7 21.9 23 Glycogen 20192.1 21013.9 18363.1 17847.2 9184.9 15900.8 19296.4 14284.1 27766.5 20989.4 10823.1 16101.4 11947.2 22048.4 21281 23302.2 18573 20576.5 16346.4 38088.3 29014.9 28575.9 24833.7 25842.4 29010.6 31035.5 28000.8 15206.9 21313 Guanosine 0.4 0.9 0.5 0.7 0.5 0.8 0.6 0.6 0.7 0.1 0.2 0.5 0.1 0.2 0.5 0.2 0.5 0.8 0.4 Guanosine diphosphate 4.6 4.8 4.2 3.9 3.6 3.5 5.8 4.6 4.8 4 3.4 5.8 4.2 5.4 4 6.1 3 4.3 4.5 2.4 2.3 4.2 3.2 2.7 2.1 2.7 2.8 2 2.3 Histidine 3.7 3.3 1.8 1.7 2.5 2.8 2.7 1.9 3.2 1 2 1.7 1.9 2.1 1.8 1.6 1.3 1.1 1 1.6 1.9 2 1.4 2.5 1.9 0.8 1.3 Hydroxyacetone 6.5 8.9 6.6 7 4.5 3.2 7.7 6 8.1 8.7 3.7 4.2 3.6 3.9 3.3 3.3 3.2 5.1 2.9 5 5.4 6 3.4 4.6 4.2 5.3 5.9 3.9 4.7 Inosine 1.3 0.8 1.8 1.5 2.5 1.8 1.7 2.2 1.9 2.6 2 2.2 3.7 2.8 1.8 4.2 2.5 3.5 1.4 0.4 0.6 0.7 0.6 0.5 0.7 0.6 0.4 0.4 0.5 Inosine triphosphate 6.7 4.3 3 2.7 4.6 4.3 5.4 5 5.8 5.2 5.9 5.9 3.9 4 4.5 3.2 2.4 4.5 4.3 4.7 4 3.8 2.7 Isoleucine 4.7 4.6 4.3 3.9 5.3 4.1 5 5.1 4.8 4 3.5 2.8 2.4 3.5 3.3 3.4 3.5 2.6 2.4 4 4 4.6 3.3 4.4 4.3 4.6 4.2 3.5 3.5 Lactate 35.2 41.5 19.4 18 24.3 21.4 29 20.7 33.7 15.2 11.3 24.4 17 19.1 15.6 26.9 11.3 13.8 11.6 13.5 13.3 15.4 14.9 20.8 16.1 32.5 21.4 12.4 12.2 Leucine 7.3 8.5 8.4 6.5 7 6.9 7.5 7 8 6.9 6.2 6.2 3.9 5.1 9.2 4.7 8 6.7 7.8 4.6 4.4 5.6 3.5 4 5.1 4.2 4.1 4 3.8 Lysine 8.6 9.2 7.4 6 17.1 12.6 9.6 8.7 7.1 6.1 9.9 11.1 5.09 8.02 11.1 8.2 8.1 9.9 7.4 6.9 6.9 11.8 6.7 6.8 8.5 6.8 8.9 5.6 7.8 Methanol 13.4 12.2 14.1 12.2 11.9 12 14.3 13 12.6 21.4 13.9 11.5 14.3 13.8 11.4 15.8 12.9 11.1 12.4 12.3 12.1 10.1 11.1 12.6 14.7 12 12.7 12.7 10.5 N-Acetyl-L-alanine 10.1 10.4 6.7 6.2 6.2 5.6 7.1 8.5 11.5 8.3 3.8 3.5 4.2 3.9 4.1 4.4 4.9 5.1 4.3 4.8 6.2 4.6 4.6 5 4.2 4.3 5.9 4.8 5.5 N-Acetylputrescine 47.5 38.9 27.3 19.7 48.4 39.8 34.1 28.3 44.9 19.3 21.9 20 7.3 11.3 31.4 12 24.3 25.8 22.6 32.7 27.2 28.7 18.6 27 27.6 23 18.9 26.8 30 NAD+ 15.6 12.4 14.5 15.9 16.4 17.4 14.7 12.5 11.8 12.8 22.5 21.5 17.3 17.5 20.6 20.5 21.2 23.8 23.5 16.7 19.1 17.4 15.4 14.3 14.2 13 13.1 15 16.4 NADH 3.2 4 3.3 4.5 4.5 3.3 5.5 6.1 6.3 5.6 0.4 3.8 3.6 2.2 1.6 3.6 1.8 1.3 1.8 3.4 2.5 3.8 3.6 2.5 2.2 2.7 2 4.3 4.2 NADP+ 5 5.2 5.7 6.1 7.3 6.1 6.5 4.9 5.1 5 5.4 6.3 5.3 6.4 4.5 6.3 6.2 6.1 6.5 5.1 5.7 5 4.5 4.5 4.4 3.8 3.7 5.3 5.4 Nicotinate 5.2 4.4 4.6 4.1 5 5.4 5.1 4.6 5.4 4.3 3.2 5.4 4.2 O-Acetylcholine 1.1 0.8 1.3 1 0.8 0.8 0.8 0.9 1.1 0.5 0.5 0.3 0.3 0.3 0.9 0.3 0.7 0.5 0.5 0.7 0.5 0.7 0.3 0.7 0.7 0.5 0.7 0.8 0.8 Phenylalanine 1.6 1.6 1.6 1.6 2.5 2.4 1.8 1.7 1.3 1.4 1.2 1.5 0.9 1.9 1.9 1.6 2 1.6 1.5 1.1 1.3 1 1.1 0.8 1.1 1 Phosphoenolpyruvic acid 45.8 38.3 62 53.2 72.8 70 47.1 40.9 42.7 41.9 75.9 60.4 42.9 48.4 50.2 46.7 70.7 66.9 78.5 80.7 65 45.9 35.3 34.7 55.6 32.2 23.2 88.4 72.8 Proline 9.5 6 3.5 6.8 2.7 3.5 5.3 11.9 6.2 6.3 3.6 7.8 13.6 14.2 11.4 17.7 9.2 5.4 7.7 11.6 4.3 12.9 8.2 13.4 13.5 16.2 16.5 9.6 6.4 Putrescine 25.8 20.8 15.6 13.8 15.2 17.8 13.6 16.5 28.7 18.2 13.1 18 19.1 23 15.9 25.7 18.4 19.3 19.2 8.6 8.4 9.4 8.3 9.7 6.8 13.5 10.1 7.4 6.9 Pyruvate 4.4 3.3 4.4 6.2 5 4.3 6.9 7.4 4.7 4.5 5.4 5.7 3.8 3.7 4.5 3.8 5.2 5.5 5.3 2.5 3.1 4 2.9 2.7 2.4 2.4 2.6 2.9 3.6 sn-Glycero-3-phosphocholine 5.2 5.7 2.8 2.5 5.7 4.1 3.6 3.8 4.9 2.2 2.4 1.8 0.9 1.2 4 1.5 2.9 2.2 2.4 3.6 2.8 3.3 2 3.2 3.1 2.6 2.3 2.9 3.2 Succinate 150.3 187 113 97.9 100.1 104.5 99.3 102.6 202 121.3 80.5 82.6 118.3 94.7 87.8 135.6 92.5 95 71.2 86.9 80.9 90.5 76.2 85.4 70.8 115.9 79 73.6 90.5 Threonine 25.5 24.7 21.9 23.6 23.6 21.6 29 23.5 30.7 15.2 8.5 17.3 33 70.1 11.1 47.8 13.8 11.7 8.8 10.5 10 11.6 10.7 13.4 12.5 11.2 14.2 10.8 9.7 Thymidine 5'-triphosphate 7.2 9.4 1.9 10.1 12.4 15.2 22.6 19.5 20.2 24.9 19 12 2.4 3.6 10.5 10.5 12.5 9.6 8.3 7.3 Tyrosine 6.4 5.8 3.7 4 6.5 5.2 5.2 4.3 4.6 3 3.1 3.7 3.1 4.8 4 4 3.9 3.1 3.1 2.6 2.5 2.2 2 2.7 2.8 2.6 2.2 2.1 2.7 UDP-glucose 19.7 18.3 19 15 21.3 19.4 19.8 14 23.1 18.8 11.9 18.6 18.1 23.2 22.4 22.1 14.7 15.7 15.7 4.6 8.5 8.9 8.2 9.9 5.4 13.7 10.9 4.5 7.2 UMP 16.5 12.9 6.6 9.6 8.6 6.4 8.7 8.4 13 8.7 8.7 6.4 5.1 8.9 16.5 9 13.2 15 8.5 12.3 13.4 13.3 10.1 14.4 13.2 15.2 14.2 10.3 12.1 Uracil 8.2 1.5 9.3 1.9 11.7 6.4 4.1 11.1 9.8 3.8 2.2 3.9 6.4 5.4 5.4 8.6 10.3 Uridine 5'-diphosphate 39.5 37.3 18.6 36.2 18.3 30.1 19.3 37.7 27.8 26.9 27.2 30.6 26.6 36.4 32.7 32 37 41.2 33.9 40.9 54.3 34.6 35.3 29.6 27 32.4 43.8 37.3 Valine 31.2 31.3 24.9 25.6 73.3 33.8 33.8 38.9 30 11.7 7.5 4.9 3.3 4 16.6 4.1 13.7 6.6 11.6 107.3 121.3 113.7 71 87.8 107.8 69.4 63.4 110.8 125.3 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name HMDB PubChem ChEBI KEGG 2-Heptanone HMDB0003671 8051 5672 C08380 3-Hydroxy-3-methylglutarate HMDB0000355 1662 16831 C03761 4-Aminobutyrate HMDB0000112 119 16865 C00334 Acetate HMDB0000042 176 15366 C00033 Acetoin HMDB0003243 179 15688 C00466 Acetone HMDB0001659 180 15347 C00207 ADP HMDB0001341 6022 16761 C00008 Alanine NA NA NA C01401 AMP HMDB0000045 6083 16027 C00020 Arginine HMDB0000517 6322 16467 C00062 Aspartate HMDB0000191 5960 17053 C00049 ATP HMDB0000538 5957 15422 C00002 Choline HMDB0000097 305 15354 C00114 Citrate HMDB0000094 311 30769 C00158 Coenzyme A HMDB0001423 87642 15346 C00010 Cytidine HMDB0000089 6175 17562 C00475 Cytidine monophosphate HMDB0000095 6131 17361 C00055 dCMP HMDB0001202 13945 15918 C00239 Ethanol HMDB0000108 702 16236 C00469 Formate HMDB0304356 283 15740 NA Fumarate HMDB0000134 444972 18012 C00122 Glucose HMDB0000122 5793 15903 C00031 Glucose-1-phosphate HMDB0001586 65533 29042 C00103 Glucose-6-phosphate HMDB0001401 5958 4170 C00092 Glutamate HMDB0000148 33032 16015 C00025 Glutamine HMDB0000641 5961 18050 C00064 Glutathione HMDB0000125 124886 16856 C00051 Glycine HMDB0000123 750 15428 C00037 Glycogen HMDB0000757 439177 28087 C00182 Guanosine HMDB0000133 6802 16750 C00387 Guanosine diphosphate HMDB0001201 8977 17552 C00035 Histidine HMDB0000177 6274 15971 C00135 Hydroxyacetone HMDB0006961 8299 27957 C05235 Inosine HMDB0000195 6021 17596 C00294 Inosine triphosphate HMDB0000189 8583 16039 C00081 Isoleucine HMDB0000172 6306 17191 C00407 Lactate HMDB0000190 107689 422 C00186 Leucine HMDB0000687 6106 15603 C00123 Lysine HMDB0000182 5962 18019 C00047 Methanol HMDB0001875 887 17790 C00132 N-Acetyl-L-alanine HMDB0000766 88064 40992 NA N-Acetylputrescine HMDB0002064 122356 17768 C02714 NAD+ HMDB0000902 5892 44215 C00003 NADH HMDB0001487 439153 16908 C00004 NADP+ HMDB0000217 5885 44409 C00006 Nicotinate HMDB0001488 938 15940 C00253 O-Acetylcholine HMDB0000895 187 15355 C01996 Phenylalanine HMDB0000159 6140 17295 C00079 Phosphoenolpyruvic acid HMDB0000263 1005 44897 C00074 Proline HMDB0000162 145742 17203 C00148 Putrescine HMDB0001414 1045 17148 C00134 Pyruvate HMDB0000243 1060 32816 C00022 sn-Glycero-3-phosphocholine HMDB0000086 657272 16870 C00670 Succinate HMDB0000254 1110 15741 C00042 Threonine HMDB0000167 6288 16857 C00188 Thymidine 5'-triphosphate HMDB0001342 64968 18077 C00459 Tyrosine HMDB0000158 6057 17895 C00082 UDP-glucose HMDB0000286 8629 46229 C00029 UMP HMDB0000288 6030 16695 C00105 Uracil HMDB0000300 1174 17568 C00106 Uridine 5'-diphosphate HMDB0000295 6031 17659 C00015 Valine HMDB0000883 6287 16414 C00183 METABOLITES_END #END