#METABOLOMICS WORKBENCH bouranij_20221027_105836 DATATRACK_ID:3539 STUDY_ID:ST002338 ANALYSIS_ID:AN003821 PROJECT_ID:PR001501 VERSION 1 CREATED_ON October 28, 2022, 9:24 am #PROJECT PR:PROJECT_TITLE Interplay Between Cruciferous Vegetables and the Gut Microbiome: A Multi-Omic PR:PROJECT_TITLE Approach PR:PROJECT_TYPE Untargeted Metabolomics PR:PROJECT_SUMMARY Untargeted metabolomics analysis of human fecal cultures following incubation PR:PROJECT_SUMMARY with in vitro digested cruciferous vegetables. The goal of this study was to PR:PROJECT_SUMMARY identify microbial- and plant-dervied metabolites of cruciferous vegetables. An PR:PROJECT_SUMMARY ex vivo fecal incubation system reflecting the digestive metabolome was used. PR:PROJECT_SUMMARY Accompanying 16S data can be retrieved in the NCBI SRA under BioProject PR:PROJECT_SUMMARY PRJNA895102. PR:INSTITUTE Oregon State University PR:DEPARTMENT Linus Pauling Institute PR:LABORATORY Emily Ho PR:LAST_NAME Bouranis PR:FIRST_NAME John PR:ADDRESS 371 Linus Pauling Science Center, 2900 SW Campus Way, Corvallis, OR, 97331, USA PR:EMAIL bouranij@oregonstate.edu PR:PHONE 5417375049 PR:FUNDING_SOURCE United States Department of Agriculture National Institute of Food and PR:FUNDING_SOURCE Agriculture (NI-FA-2020-67001-31214; NIFA-2022-67011-36576), National Institutes PR:FUNDING_SOURCE of Health (P30ES030287; S10RR027878), Oregon Agricultural Experimental Station PR:FUNDING_SOURCE (W4002; OR00735) #STUDY ST:STUDY_TITLE Interplay Between Cruciferous Vegetables and the Gut Microbiome: A Multi-Omic ST:STUDY_TITLE Approach ST:STUDY_TYPE Ex Vivo Fecal Incubation ST:STUDY_SUMMARY Cruciferous vegetable consumption has been associated with a decreased risk of ST:STUDY_SUMMARY multiple types of cancers, thus presenting a cost-effective, non-pharmacological ST:STUDY_SUMMARY approach to cancer prevention through dietary intervention. Broccoli sprouts and ST:STUDY_SUMMARY Brussels sprouts are among the leading cruciferous vegetables under study and ST:STUDY_SUMMARY contain some similar and some distinct phytochemicals which can activate ST:STUDY_SUMMARY different, but complementary, mechanisms to promote health. While the ST:STUDY_SUMMARY cancer-preventative effects of cruciferous vegetables are typically attributed ST:STUDY_SUMMARY to glucosinolates and their metabolic products, isothiocyanates and indoles, ST:STUDY_SUMMARY other components of cruciferous vegetables could play a synergistic role in ST:STUDY_SUMMARY conferring cancer-protective and health promoting effects. Additionally, ST:STUDY_SUMMARY metabolism of phytochemicals from cruciferous vegetables by the gut microbiome ST:STUDY_SUMMARY could further lead to the production, inactivation, or clearance of bioactive ST:STUDY_SUMMARY dietary components. The gut microbiome is essential to the production of ST:STUDY_SUMMARY bioactive compounds from various food sources. For example, with soy isoflavones ST:STUDY_SUMMARY and pomegranate urolithins, the presence or absence of specific microbial taxa ST:STUDY_SUMMARY directly dictates which metabolites are produced (resulting in a metabotype). A ST:STUDY_SUMMARY similar paradigm could be extended to cruciferous vegetables in which the gut ST:STUDY_SUMMARY microbiome may play an important role in driving inter-individual metabolism of ST:STUDY_SUMMARY glucosinolates and isothiocyanates. We recently reported (Bouranis et. al, 2021, ST:STUDY_SUMMARY Nutrients) that the gut microbiome composition can influence production of ST:STUDY_SUMMARY glucosinolate-derived nitriles from cruciferous vegetables, showing that the ST:STUDY_SUMMARY presence or absence of specific microbes can influence the abundance of a single ST:STUDY_SUMMARY metabolite. Thus, we sought to take an untargeted approach to investigate other ST:STUDY_SUMMARY phytochemicals from cruciferous vegetables which the gut microbiome could play a ST:STUDY_SUMMARY role in generating. To investigate plant- and microbe-derived metabolites of ST:STUDY_SUMMARY cruciferous vegetable digestion and capture information about the microbiome, we ST:STUDY_SUMMARY utilized an ex vivo fecal incubation system. Broccoli sprouts and Brussels ST:STUDY_SUMMARY sprouts were in vitro digested using an oral, gastric, and intestinal phase. For ST:STUDY_SUMMARY fecal bacterial cultivation a 20% fecal slurry (w/v) was made from fecal ST:STUDY_SUMMARY material from 10 healthy volunteers (6 female, and 4 male, age 17-51, Lee ST:STUDY_SUMMARY Biosolutions) and sterile PBS (0.1 M pH 7). 500 µL of fecal slurry was mixed ST:STUDY_SUMMARY with 10 mL of Brain Heart Infusion Broth (BHI) with hemin and vitamin K, per the ST:STUDY_SUMMARY manufacturer’s recommendation, and either 500 µl of filter sterilized in ST:STUDY_SUMMARY vitro digested broccoli sprouts (Broc), 500 µL of filter sterilized in vitro ST:STUDY_SUMMARY digested Brussels sprouts (Brus), 500 µL of Broc and 500 µL of Brus were added ST:STUDY_SUMMARY (Combo) or a negative control in vitro digestion (NC). NC contained reverse ST:STUDY_SUMMARY osmosis water, equivalent in volume to the water content of broccoli sprouts and ST:STUDY_SUMMARY underwent the same in vitro digestion procedure as described above with the same ST:STUDY_SUMMARY enzymes, chemicals and equipment. Broc and Brus digests were scaled to be ST:STUDY_SUMMARY equivalent in concentration to a human consuming ½ cup of broccoli or Brussels ST:STUDY_SUMMARY sprouts, or in the case of the combination, ½ cup of broccoli sprouts and ½ ST:STUDY_SUMMARY cup of Brussels sprouts. This combination was included as Broc and Brus contain ST:STUDY_SUMMARY many similar but also some distinct phytochemicals and thus by combining the ST:STUDY_SUMMARY vegetables we increased the dose and broadened the range of phytochemicals from ST:STUDY_SUMMARY cruciferous vegetables which can be achieved in the kitchen as a mixed vegetable ST:STUDY_SUMMARY dish. Fecal cultures were incubated at 37°C for 24 h in anaerobic conditions. ST:INSTITUTE Oregon State University ST:DEPARTMENT Linus Pauling Institute ST:LABORATORY Emily Ho ST:LAST_NAME Bouranis ST:FIRST_NAME John ST:ADDRESS 371 Linus Pauling Science Center, 2900 SW Campus Way, Corvallis, OR, 97331, USA ST:EMAIL bouranij@oregonstate.edu ST:PHONE 5417375049 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 10 ST:NUM_MALES 4 ST:NUM_FEMALES 6 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 17-51 SU:GENDER Male and female SU:CELL_BIOSOURCE_OR_SUPPLIER Lee Biosolutions SU:SUBJECT_COMMENTS Human fecal cultures #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 T6291 ms_51 treatment:combo | group:E_Type 16s_sample=80; birth_year=1985; biological_sex=female; RAW_FILE_NAME=neg_51.mzXML SUBJECT_SAMPLE_FACTORS T6291 ms_50 treatment:brus | group:E_Type 16s_sample=79; birth_year=1985; biological_sex=female; RAW_FILE_NAME=neg_50.mzXML SUBJECT_SAMPLE_FACTORS T6291 ms_49 treatment:broc | group:E_Type 16s_sample=78; birth_year=1985; biological_sex=female; RAW_FILE_NAME=neg_49.mzXML SUBJECT_SAMPLE_FACTORS T6291 ms_48 treatment:NC | group:E_Type 16s_sample=77; birth_year=1985; biological_sex=female; RAW_FILE_NAME=neg_48.mzXML SUBJECT_SAMPLE_FACTORS T1995 ms_47 treatment:combo | group:C_Type 16s_sample=75; birth_year=1986; biological_sex=male; RAW_FILE_NAME=neg_47.mzXML SUBJECT_SAMPLE_FACTORS T1995 ms_46 treatment:brus | group:C_Type 16s_sample=74; birth_year=1986; biological_sex=male; RAW_FILE_NAME=neg_46.mzXML SUBJECT_SAMPLE_FACTORS T1995 ms_45 treatment:broc | group:C_Type 16s_sample=73; birth_year=1986; biological_sex=male; RAW_FILE_NAME=neg_45.mzXML SUBJECT_SAMPLE_FACTORS T1995 ms_44 treatment:NC | group:C_Type 16s_sample=72; birth_year=1986; biological_sex=male; RAW_FILE_NAME=neg_44.mzXML SUBJECT_SAMPLE_FACTORS T5717 ms_43 treatment:combo | group:C_Type 16s_sample=70; birth_year=1978; biological_sex=female; RAW_FILE_NAME=neg_43.mzXML SUBJECT_SAMPLE_FACTORS T5717 ms_42 treatment:brus | group:C_Type 16s_sample=69; birth_year=1978; biological_sex=female; RAW_FILE_NAME=neg_42.mzXML SUBJECT_SAMPLE_FACTORS T5717 ms_41 treatment:broc | group:C_Type 16s_sample=68; birth_year=1978; biological_sex=female; RAW_FILE_NAME=neg_41.mzXML SUBJECT_SAMPLE_FACTORS T5717 ms_40 treatment:NC | group:C_Type 16s_sample=67; birth_year=1978; biological_sex=female; RAW_FILE_NAME=neg_40.mzXML SUBJECT_SAMPLE_FACTORS T5631 ms_39 treatment:combo | group:E_Type 16s_sample=65; birth_year=1999; biological_sex=male; RAW_FILE_NAME=neg_39.mzXML SUBJECT_SAMPLE_FACTORS T5631 ms_38 treatment:brus | group:E_Type 16s_sample=64; birth_year=1999; biological_sex=male; RAW_FILE_NAME=neg_38.mzXML SUBJECT_SAMPLE_FACTORS T5631 ms_37 treatment:broc | group:E_Type 16s_sample=63; birth_year=1999; biological_sex=male; RAW_FILE_NAME=neg_37.mzXML SUBJECT_SAMPLE_FACTORS T5631 ms_36 treatment:NC | group:E_Type 16s_sample=62; birth_year=1999; biological_sex=male; RAW_FILE_NAME=neg_36.mzXML SUBJECT_SAMPLE_FACTORS T5854 ms_35 treatment:combo | group:C_Type 16s_sample=60; birth_year=1984; biological_sex=female; RAW_FILE_NAME=neg_35.mzXML SUBJECT_SAMPLE_FACTORS T5854 ms_34 treatment:brus | group:C_Type 16s_sample=59; birth_year=1984; biological_sex=female; RAW_FILE_NAME=neg_34.mzXML SUBJECT_SAMPLE_FACTORS T5854 ms_33 treatment:broc | group:C_Type 16s_sample=58; birth_year=1984; biological_sex=female; RAW_FILE_NAME=neg_33.mzXML SUBJECT_SAMPLE_FACTORS T5854 ms_32 treatment:NC | group:C_Type 16s_sample=57; birth_year=1984; biological_sex=female; RAW_FILE_NAME=neg_32.mzXML SUBJECT_SAMPLE_FACTORS T6260 ms_31 treatment:combo | group:E_Type 16s_sample=55; birth_year=1973; biological_sex=female; RAW_FILE_NAME=neg_31.mzXML SUBJECT_SAMPLE_FACTORS T6260 ms_30 treatment:brus | group:E_Type 16s_sample=54; birth_year=1973; biological_sex=female; RAW_FILE_NAME=neg_30.mzXML SUBJECT_SAMPLE_FACTORS T6260 ms_29 treatment:broc | group:E_Type 16s_sample=53; birth_year=1973; biological_sex=female; RAW_FILE_NAME=neg_29.mzXML SUBJECT_SAMPLE_FACTORS T6260 ms_28 treatment:NC | group:E_Type 16s_sample=52; birth_year=1973; biological_sex=female; RAW_FILE_NAME=neg_28.mzXML SUBJECT_SAMPLE_FACTORS T5632 ms_27 treatment:combo | group:E_Type 16s_sample=50; birth_year=2001; biological_sex=male; RAW_FILE_NAME=neg_27.mzXML SUBJECT_SAMPLE_FACTORS T5632 ms_26 treatment:brus | group:E_Type 16s_sample=49; birth_year=2001; biological_sex=male; RAW_FILE_NAME=neg_26.mzXML SUBJECT_SAMPLE_FACTORS T5632 ms_25 treatment:broc | group:E_Type 16s_sample=48; birth_year=2001; biological_sex=male; RAW_FILE_NAME=neg_25.mzXML SUBJECT_SAMPLE_FACTORS T5632 ms_24 treatment:NC | group:E_Type 16s_sample=47; birth_year=2001; biological_sex=male; RAW_FILE_NAME=neg_24.mzXML SUBJECT_SAMPLE_FACTORS T5627 ms_23 treatment:combo | group:C_Type 16s_sample=45; birth_year=1988; biological_sex=female; RAW_FILE_NAME=neg_23.mzXML SUBJECT_SAMPLE_FACTORS T5627 ms_22 treatment:brus | group:C_Type 16s_sample=44; birth_year=1988; biological_sex=female; RAW_FILE_NAME=neg_22.mzXML SUBJECT_SAMPLE_FACTORS T5627 ms_21 treatment:broc | group:C_Type 16s_sample=43; birth_year=1988; biological_sex=female; RAW_FILE_NAME=neg_21.mzXML SUBJECT_SAMPLE_FACTORS T5627 ms_20 treatment:NC | group:C_Type 16s_sample=42; birth_year=1988; biological_sex=female; RAW_FILE_NAME=neg_20.mzXML SUBJECT_SAMPLE_FACTORS T6382 ms_19 treatment:combo | group:C_Type 16s_sample=40; birth_year=1967; biological_sex=male; RAW_FILE_NAME=neg_19.mzXML SUBJECT_SAMPLE_FACTORS T6382 ms_18 treatment:brus | group:C_Type 16s_sample=39; birth_year=1967; biological_sex=male; RAW_FILE_NAME=neg_18.mzXML SUBJECT_SAMPLE_FACTORS T6382 ms_17 treatment:broc | group:C_Type 16s_sample=38; birth_year=1967; biological_sex=male; RAW_FILE_NAME=neg_17.mzXML SUBJECT_SAMPLE_FACTORS T6382 ms_16 treatment:NC | group:C_Type 16s_sample=37; birth_year=1967; biological_sex=male; RAW_FILE_NAME=neg_16.mzXML SUBJECT_SAMPLE_FACTORS T4669 ms_15 treatment:combo | group:E_Type 16s_sample=35; birth_year=1967; biological_sex=female; RAW_FILE_NAME=neg_15.mzXML SUBJECT_SAMPLE_FACTORS T4669 ms_14 treatment:brus | group:E_Type 16s_sample=34; birth_year=1967; biological_sex=female; RAW_FILE_NAME=neg_14.mzXML SUBJECT_SAMPLE_FACTORS T4669 ms_13 treatment:broc | group:E_Type 16s_sample=33; birth_year=1967; biological_sex=female; RAW_FILE_NAME=neg_13.mzXML SUBJECT_SAMPLE_FACTORS T4669 ms_12 treatment:NC | group:E_Type 16s_sample=32; birth_year=1967; biological_sex=female; RAW_FILE_NAME=neg_12.mzXML #COLLECTION CO:COLLECTION_SUMMARY Fecal culture medium was then vortexed, sampled, centrifuged (13,000 g, 10 CO:COLLECTION_SUMMARY min) and supernatants frozen in liquid nitrogen. CO:SAMPLE_TYPE Feces #TREATMENT TR:TREATMENT_SUMMARY Broccoli sprouts and Brussels sprouts were in vitro digested using an oral, TR:TREATMENT_SUMMARY gastric, and intestinal phase. For fecal bacterial cultivation a 20% fecal TR:TREATMENT_SUMMARY slurry (w/v) was made from fecal material from 10 healthy volunteers (6 female, TR:TREATMENT_SUMMARY and 4 male, age 17-51, Lee Biosolutions) and sterile PBS (0.1 M pH 7). 500 µL TR:TREATMENT_SUMMARY of fecal slurry was mixed with 10 mL of Brain Heart Infusion Broth (BHI) with TR:TREATMENT_SUMMARY hemin and vitamin K, per the manufacturer’s recommendation, and either 500 µl TR:TREATMENT_SUMMARY of filter sterilized in vitro digested broccoli sprouts (Broc), 500 µL of TR:TREATMENT_SUMMARY filter sterilized in vitro digested Brussels sprouts (Brus), 500 µL of Broc and TR:TREATMENT_SUMMARY 500 µL of Brus were added (Combo) or a negative control in vitro digestion TR:TREATMENT_SUMMARY (NC). NC contained reverse osmosis water, equivalent in volume to the water TR:TREATMENT_SUMMARY content of broccoli sprouts and underwent the same in vitro digestion procedure TR:TREATMENT_SUMMARY as described above with the same enzymes, chemicals and equipment. Broc and Brus TR:TREATMENT_SUMMARY digests were scaled to be equivalent in concentration to a human consuming ½ TR:TREATMENT_SUMMARY cup of broccoli or Brussels sprouts, or in the case of the combination, ½ cup TR:TREATMENT_SUMMARY of broccoli sprouts and ½ cup of Brussels sprouts. This combination was TR:TREATMENT_SUMMARY included as Broc and Brus contain many similar but also some distinct TR:TREATMENT_SUMMARY phytochemicals and thus by combining the vegetables we increased the dose and TR:TREATMENT_SUMMARY broadened the range of phytochemicals from cruciferous vegetables which can be TR:TREATMENT_SUMMARY achieved in the kitchen as a mixed vegetable dish. Fecal cultures were incubated TR:TREATMENT_SUMMARY at 37°C for 24 h in anaerobic conditions #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites from fecal culture medium were extracted (100 μL culture/100 μL SP:SAMPLEPREP_SUMMARY ice cold 80:20, v/v, methanol:water), mixed vigorously, and clarified by SP:SAMPLEPREP_SUMMARY centrifugation (13,000× g for 10 min). The supernatants were further diluted SP:SAMPLEPREP_SUMMARY 1:10 (v/v) with ice cold 80:20 methanol:water (v/v) and transferred to mass SP:SAMPLEPREP_SUMMARY spectrometry (MS) vials #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Ultra-high pressure liquid chromatography (UPLC) was performed on a Shimadzu CH:CHROMATOGRAPHY_SUMMARY Nexera™ system (Shimadzu, Columbia, MD) coupled to a quadrupole time-of-flight CH:CHROMATOGRAPHY_SUMMARY mass spectrometer (AB SCIEX TripleTOF 5600). Chromatographic separations were CH:CHROMATOGRAPHY_SUMMARY conducted on an Inertsil1 Phenyl-3 column (4.6 × 150 mm, GL Sciences, Torrance, CH:CHROMATOGRAPHY_SUMMARY CA). Elution was achieved using a binary gradient employing as solvent A water, CH:CHROMATOGRAPHY_SUMMARY and solvent B methanol, both containing 0.1% formic acid (v/ v), as described CH:CHROMATOGRAPHY_SUMMARY previously. The gradient started with 5% Band was held for 1 min at 5% B, CH:CHROMATOGRAPHY_SUMMARY followed by a 11-min linear gradient from 5% to 30% B. The gradient was CH:CHROMATOGRAPHY_SUMMARY increased linearly to 100% B at 23 min, held for 5 min at 100% B and, finally, CH:CHROMATOGRAPHY_SUMMARY stepped back to 5% B to equilibrate the column. The flow rate was 0.4 mL/min. CH:CHROMATOGRAPHY_SUMMARY The auto-sampler temperature was held at 10 ̊C, the column oven temperature at CH:CHROMATOGRAPHY_SUMMARY 50 ̊C, and the injection volume was 5 μL. Time-of-flight (TOF) mass CH:CHROMATOGRAPHY_SUMMARY spectrometry (MS) was operated with an acquisition time of 0.25 s and a scan CH:CHROMATOGRAPHY_SUMMARY range of 70–1200 Da. Tandem mass spectrometry (MS/MS) acquisition was CH:CHROMATOGRAPHY_SUMMARY performed with collision energy set at 35 V and collision energy spread of 15 V. CH:CHROMATOGRAPHY_SUMMARY Each MS/MS scan had an accumulation time of 0.17 sand a range of 50–1250 Da CH:CHROMATOGRAPHY_SUMMARY using information-dependent MS/MS acquisition (IDA). Ion source gas 1and 2and CH:CHROMATOGRAPHY_SUMMARY curtain gas (all nitrogen) were set at 50, 40, and 25, respectively. The source CH:CHROMATOGRAPHY_SUMMARY temperature was set at 500 ̊C and the ion spray voltage at 4.5 kV in positive CH:CHROMATOGRAPHY_SUMMARY ion mode. The mass calibration was automatically performed every 2 injections CH:CHROMATOGRAPHY_SUMMARY using a calibration solution (AB SCIEX) via a calibration delivery system (CDS). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera CH:COLUMN_NAME GL Sciences Inertsil1 Phenyl-3 (4.6 x 150mm) #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Oregon State Mass Spectrometry Center #MS MS:INSTRUMENT_NAME ABI Sciex 5600 TripleTOF MS:INSTRUMENT_TYPE Triple TOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The samples were randomized, auto-calibration was performed every two samples, MS:MS_COMMENTS and a quality control sample, composed of a pooled aliquot from each sample, was MS:MS_COMMENTS analyzed every 10 samples. MS/MS information was obtained for all samples using MS:MS_COMMENTS information dependent acquisition (IDA), while sequential window acquisition of MS:MS_COMMENTS all theoretical spectra (SWATH) was performed only on quality control samples. MS:MS_COMMENTS Spectral data were processed using Progenesis QI (NonLinear Dynamics v2.4). Peak MS:MS_COMMENTS deconvolution for [M + H]+, [M + Na]+, and [M + NH4]+ adducts in positive MS:MS_COMMENTS ionization mode, and [M−H]-, [M + FA-H]-, and [M−H2O−H]- in negative MS:MS_COMMENTS ionization mode was performed in Progenesis QI. Feature intensities were MS:MS_COMMENTS normalized in Progenesis QI across samples by total ion current of all features. MS:MS_RESULTS_FILE ST002338_AN003821_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END