#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