#METABOLOMICS WORKBENCH lixinwei_20240923_193010 DATATRACK_ID:5215 STUDY_ID:ST003505 ANALYSIS_ID:AN005753 PROJECT_ID:PR002151 VERSION 1 CREATED_ON September 30, 2024, 9:23 am #PROJECT PR:PROJECT_TITLE Uncovering the de novo synthesis of polyamines in gut microbiome using stable PR:PROJECT_TITLE isotope resolved metabolomics PR:PROJECT_TYPE metabolomics PR:PROJECT_SUMMARY Using 13C-inulin as a tracer, we tracked the biosynthesis of polyamines in the PR:PROJECT_SUMMARY human and mouse fecal microbiome. Additionally, single-strain SIRM analyses was PR:PROJECT_SUMMARY used to explore functional gut microbes. Liquid chromatography-high resolution PR:PROJECT_SUMMARY mass spectrometry analysis revealed distinct 13C enrichment profiles for PR:PROJECT_SUMMARY polyamines. SIRM analyses were performed using a Q-Exactive HF mass PR:PROJECT_SUMMARY spectrometer, equipped with an Ion Max API source and a HESI II probe, and were PR:PROJECT_SUMMARY coupled to a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific). The PR:PROJECT_SUMMARY findings revealed a novel de novo SPD biosynthesis pathway in the human gut PR:PROJECT_SUMMARY microbiome, and Bacteroides (including B.fragilis and B.thetaiotaomicron) PR:PROJECT_SUMMARY contributed to the biosynthesis of polyamines, underscoring the importance of PR:PROJECT_SUMMARY polyamine bioanalysis in aligning gut microbial functions to host intestinal PR:PROJECT_SUMMARY health. PR:INSTITUTE Soochow University PR:LAST_NAME li PR:FIRST_NAME xinwei PR:ADDRESS 199 Renai Road, Xietang Street, Suzhou, Suzhou, Jiangsu Province, 215031, China PR:EMAIL lxw9911117@163.com PR:PHONE 19971871675 #STUDY ST:STUDY_TITLE Investigation of polyamine biosynthesis and metabolism in gut microbiome by ST:STUDY_TITLE stale isotope resolved metabolomics ST:STUDY_SUMMARY Polyamines are important gut microbial metabolites known to affect host organs, ST:STUDY_SUMMARY yet the mechanisms behind their microbial production remain poorly understood. ST:STUDY_SUMMARY In this study, we used a stable isotope-resolved metabolomic (SIRM) approach to ST:STUDY_SUMMARY track polyamine biosynthesis in the fecal microbiome. Viable microbial cells ST:STUDY_SUMMARY were extracted from fresh human and mouse feces and incubated anaerobically with ST:STUDY_SUMMARY 13C-labeled inulin (tracer). Liquid chromatography-high resolution mass ST:STUDY_SUMMARY spectrometry analysis revealed distinct 13C enrichment profiles for spermidine ST:STUDY_SUMMARY (SPD) and putrescine (PUT), with the arginine-agmatine-SPD pathway predominating ST:STUDY_SUMMARY over the well acknowledged spermidine synthase pathway (PUT aminopropylation) ST:STUDY_SUMMARY for SPD biosynthesis. Furthermore, significant species differences were observed ST:STUDY_SUMMARY in the 13C enrichments of polyamines and related metabolites between the human ST:STUDY_SUMMARY and mouse microbiome. Further investigations using single-strain SIRM analyses ST:STUDY_SUMMARY (Bacteroides) identified the key microbial genes and gut microbes responsible ST:STUDY_SUMMARY for the polyamine biosynthesis. Taken together, this study expands our ST:STUDY_SUMMARY understanding of polyamine biosynthesis in the gut microbiome and will ST:STUDY_SUMMARY facilitate the development of precision therapies to target polyamine-associated ST:STUDY_SUMMARY diseases. ST:INSTITUTE Soochow University ST:LAST_NAME xinwei ST:FIRST_NAME li ST:ADDRESS School of Pharmacy, Soochow University 1113 ST:EMAIL lxw9911117@163.com ST:PHONE 19971871675 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Human (Intestinal bacteria); Mouse (Intestinal bacteria); Bacteroides SU:SUBJECT_SPECIES (B.fragilis and B.thetaiotaomicron) #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 A1_1 Pos_13D_1 Sample source:Human feces | Treatment Condition:HILIC-HRMS RAW_FILE_NAME=Pos_13D_1 SUBJECT_SAMPLE_FACTORS A1_1 Pos_13D_2 Sample source:Human feces | Treatment Condition:HILIC-HRMS RAW_FILE_NAME=Pos_13D_2 SUBJECT_SAMPLE_FACTORS A1_1 Pos_13D_3 Sample source:Human feces | Treatment Condition:HILIC-HRMS RAW_FILE_NAME=Pos_13D_3 SUBJECT_SAMPLE_FACTORS A1_1 NGE_D_13C_1 Sample source:Human feces | Treatment Condition:HILIC-HRMS RAW_FILE_NAME=NGE_D_13C_1 SUBJECT_SAMPLE_FACTORS A1_1 NGE_D_13C_2 Sample source:Human feces | Treatment Condition:HILIC-HRMS RAW_FILE_NAME=NGE_D_13C_2 SUBJECT_SAMPLE_FACTORS A1_1 NGE_D_13C_3 Sample source:Human feces | Treatment Condition:HILIC-HRMS RAW_FILE_NAME=NGE_D_13C_3 SUBJECT_SAMPLE_FACTORS A1_2 Hcell1 Sample source:Human feces | Treatment Condition:Fmoc-derivatization-LC-HRMS RAW_FILE_NAME=Hcell1 SUBJECT_SAMPLE_FACTORS A1_2 Hcell2 Sample source:Human feces | Treatment Condition:Fmoc-derivatization-LC-HRMS RAW_FILE_NAME=Hcell2 SUBJECT_SAMPLE_FACTORS A1_2 Hcell3 Sample source:Human feces | Treatment Condition:Fmoc-derivatization-LC-HRMS RAW_FILE_NAME=Hcell3 SUBJECT_SAMPLE_FACTORS A2_2 C57cell1 Sample source:mouse feces | Treatment Condition:Fmoc-derivatization-LC-HRMS RAW_FILE_NAME=C57cell1 SUBJECT_SAMPLE_FACTORS A2_2 C57cell2 Sample source:mouse feces | Treatment Condition:Fmoc-derivatization-LC-HRMS RAW_FILE_NAME=C57cell2 SUBJECT_SAMPLE_FACTORS A2_2 C57cell3 Sample source:mouse feces | Treatment Condition:Fmoc-derivatization-LC-HRMS RAW_FILE_NAME=C57cell3 SUBJECT_SAMPLE_FACTORS A3_1 T1P-pos Sample source:Bacteroides thetaiotaomicron cells | Treatment Condition:HILIC-HRMS RAW_FILE_NAME=T1P-pos SUBJECT_SAMPLE_FACTORS A3_1 T2P-pos Sample source:Bacteroides thetaiotaomicron cells | Treatment Condition:HILIC-HRMS RAW_FILE_NAME=T2P-pos SUBJECT_SAMPLE_FACTORS A3_1 T3P-pos Sample source:Bacteroides thetaiotaomicron cells | Treatment Condition:HILIC-HRMS RAW_FILE_NAME=T3P-pos SUBJECT_SAMPLE_FACTORS A3_1 F1P-pos Sample source:Bacteroides fragilis cells | Treatment Condition:HILIC-HRMS RAW_FILE_NAME=F1P-pos SUBJECT_SAMPLE_FACTORS A3_1 F2P-pos Sample source:Bacteroides fragilis cells | Treatment Condition:HILIC-HRMS RAW_FILE_NAME=F2P-pos SUBJECT_SAMPLE_FACTORS A3_1 F3P-pos Sample source:Bacteroides fragilis cells | Treatment Condition:HILIC-HRMS RAW_FILE_NAME=F3P-pos SUBJECT_SAMPLE_FACTORS A3_2 XBPC18_13C-T1C-pos Sample source:Bacteroides thetaiotaomicron cells | Treatment Condition:Fmoc-derivatization-LC-HRMS RAW_FILE_NAME=XBPC18_13C-T1C-pos SUBJECT_SAMPLE_FACTORS A3_2 XBPC18_13C-T2C-pos Sample source:Bacteroides thetaiotaomicron cells | Treatment Condition:Fmoc-derivatization-LC-HRMS RAW_FILE_NAME=XBPC18_13C-T2C-pos SUBJECT_SAMPLE_FACTORS A3_2 XBPC18_13C-T3C-pos Sample source:Bacteroides thetaiotaomicron cells | Treatment Condition:Fmoc-derivatization-LC-HRMS RAW_FILE_NAME=XBPC18_13C-T3C-pos SUBJECT_SAMPLE_FACTORS A3_2 XBPC18_13C-F1C-pos Sample source:Bacteroides fragilis cells | Treatment Condition:Fmoc-derivatization-LC-HRMS RAW_FILE_NAME=XBPC18_13C-F1C-pos SUBJECT_SAMPLE_FACTORS A3_2 XBPC18_13C-F2C-pos Sample source:Bacteroides fragilis cells | Treatment Condition:Fmoc-derivatization-LC-HRMS RAW_FILE_NAME=XBPC18_13C-F2C-pos SUBJECT_SAMPLE_FACTORS A3_2 XBPC18_13C-F3C-pos Sample source:Bacteroides fragilis cells | Treatment Condition:Fmoc-derivatization-LC-HRMS RAW_FILE_NAME=XBPC18_13C-F3C-pos #COLLECTION CO:COLLECTION_SUMMARY A fraction of the fecal sample was collected fresh in its native state in a CO:COLLECTION_SUMMARY sterile screw-cap container. The samples were quickly transferred to an CO:COLLECTION_SUMMARY anaerobic glove bag to ensure fecal microbiome vitality. The fresh fecal samples CO:COLLECTION_SUMMARY were dissolved in the culture media and processed with a glass rods to suspend CO:COLLECTION_SUMMARY the microorganisms and particles. Then, the suspensions were transferred to an CO:COLLECTION_SUMMARY anaerobic tube and subjected to low-speed centrifugation (600 rpm, 10 min) to CO:COLLECTION_SUMMARY remove larger particles of undigested material. The supernatants were then CO:COLLECTION_SUMMARY collected and centrifuged at 3,000 rpm for 10 min to pellet the microbes. After CO:COLLECTION_SUMMARY washing the precipitated microorganisms using culture medium, the fecal CO:COLLECTION_SUMMARY microbial cells were collected by centrifugation at 3000 rpm for 10 min. CO:SAMPLE_TYPE Feces (including human and mouse); Bacteroides (including B.fragilis and CO:SAMPLE_TYPE B.thetaiotaomicron) #TREATMENT TR:TREATMENT_SUMMARY 13C-Inulin was added to each tube aseptically to achieve a final concentration TR:TREATMENT_SUMMARY of 2 g/L inulin. After incubating at 37ºC for 24 h, the samples were TR:TREATMENT_SUMMARY centrifuged and washed with fresh culture medium and centrifuged to collect the TR:TREATMENT_SUMMARY microbial cells for further analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For the analysis of polyamines, the microbial cells were quenched immediately SP:SAMPLEPREP_SUMMARY after collection using 200 μL of acetonitrile containing 0.2% formic acid. To SP:SAMPLEPREP_SUMMARY each 50 μL aliquot of the sample solution, 50 μL of carbonic acid buffer (0.5 SP:SAMPLEPREP_SUMMARY M, pH 10.2) was added, followed by 50 μL of 5 mM Fmoc-OSu solution in SP:SAMPLEPREP_SUMMARY acetonitrile. The mixture was shaken and incubated at room temperature for 15 SP:SAMPLEPREP_SUMMARY min. The derivatization reaction was quenched with 20 μL of formic acid, and SP:SAMPLEPREP_SUMMARY the sample was extracted with 500 μL of ethyl acetate. After vortexing for 2 SP:SAMPLEPREP_SUMMARY min, the sample was centrifuged and the organic phase was collected and dried SP:SAMPLEPREP_SUMMARY under a nitrogen stream. The samples were stored at −80ºC and dissolved in SP:SAMPLEPREP_SUMMARY acetonitrile: water (9:1, v/v) before analysis. For the untargeted metabolomic SP:SAMPLEPREP_SUMMARY analysis (polar metabolites), the microbial cells were quenched using 450 μL SP:SAMPLEPREP_SUMMARY cold methanol immediately after collection. 5 mL of methyl tert-butyl ether was SP:SAMPLEPREP_SUMMARY added to each tube, and phase separation was then induced by adding 1.25 mL of SP:SAMPLEPREP_SUMMARY deionized water. The samples were then vortexed briefly and centrifuged. Polar SP:SAMPLEPREP_SUMMARY fractions were collected into clean tubes and lyophilized. The dried powder was SP:SAMPLEPREP_SUMMARY stored at −80ºC and dissolved in methanol: water (8:2, v/v) before analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Fmoc-derivatization-LC-HRMS CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Q-Exactive HF CH:COLUMN_NAME Agela Technologies Venusil XBP C18 column (50 x 2.1 mm, 5 μm) CH:SOLVENT_A 100% Water; 0.1% Formic acid CH:SOLVENT_B 100% Acetonitrile CH:FLOW_GRADIENT 0–10 min, linear gradient from 10% to 90% B; 10–14 min, hold at 90% B; CH:FLOW_GRADIENT 14–14.5 min, linear gradient to 10% B; 14.5–20 min, hold at 10% B; and stop CH:FLOW_GRADIENT running at 32 min. CH:FLOW_RATE 200 μL/min CH:COLUMN_TEMPERATURE 40˚C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q-Exactive HF MS:INSTRUMENT_TYPE Q Exactive HF MS:MS_TYPE API MS:ION_MODE POSITIVE MS:MS_COMMENTS The high resolution mass spectrometer was operated in positive mode with the MS:MS_COMMENTS spray voltage set to 4.0 kV, the heated capillary was held at 350 °C, and the MS:MS_COMMENTS HESI probe was set at 325 °C. The sheath gas flow was set as 45 units, the MS:MS_COMMENTS auxiliary gas flow was set as 10 units, and the sweep gas flow was set to 1 MS:MS_COMMENTS unit. The MS data acquisition was performed in the range of 75–1000 m/z, with MS:MS_COMMENTS the resolution set at 60,000, the AGC targeted at 1e6, and the maximum injection MS:MS_COMMENTS time was set at 200 ms. The peak areas of the metabolites and their MS:MS_COMMENTS isotopologues were integrated and exported to Excel via the Thermo Scientific MS:MS_COMMENTS Xcalibur (version 4.2.47). Fractional 13C metabolite enrichment was obtained MS:MS_COMMENTS after natural abundance stripping using Escher-Trace MS:MS_COMMENTS (https://escher-trace.github.io/app/index.html). MS:MS_RESULTS_FILE ST003505_AN005753_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END