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MB Sample ID: SA200945

Local Sample ID:	ms5520-13
Subject ID:	SU002178
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Subject:

Subject ID:	SU002178
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
ms5520-13	SA200945	FL024378	Case-Low PA PI-IBS	Group

Collection:

Collection ID:	CO002171
Collection Summary:	Fecal supernatants (FSNs) were made fresh prior to each experiment. Feces from patients (0.1g) or mice (1 pellet) was added to 0.8mL of phosphate buffered saline (PBS) and subsequently homogenized with a pellet pestle for 5-10 seconds (Sigma-Aldrich, St. Louis, MO, USA). Homogenates were spun twice at 5,000 g for 10 min at 4°C and then added to a 0.22 µm Spin-X tube filter (Corning Life Sciences, Durham, NC, USA). Samples were filtered at 4°C, 10,000 g for 5 min and FSN was stored on ice until use.
Sample Type:	Feces

Treatment:

Treatment ID:	TR002190
Treatment Summary:	A total of 52 PI-IBS patients defined by Rome III criteria and 38 healthy volunteers were recruited. Those with a history of abdominal surgery (except hernia, C-section, hysterectomy, appendectomy or cholecystectomy), inflammatory bowel disease, microscopic colitis, or celiac disease were excluded. Additionally, recruited volunteers were not pregnant at the time of the study. Use of tobacco or alcohol for the duration of the study was prohibited. Following medications were prohibited 7 days prior to study participation: those affecting gastrointestinal transit, serotonergic agents, anti-cholinergic agents, antimuscarinics, narcotics, peppermint oil, antibiotics or new probiotics. Ingestion of artificial sweeteners such as SplendaTM (sucralose), Nutrasweet TM (aspartame), lactulose or mannitol was prohibited for 2 days before the start and during the study. All subjects taking part in the study were asked to complete the Hospital Anxiety and Depression Scale (HADS) and a 7-day bowel diary. All participants completed the Hospital anxiety and depression scale (HADS). PI-IBS patients also completed the Symptom Checklist-90 (SCL-90), IBS Symptom severity scale (IBS-SSS), IBS-quality of life (IBS-QoL) questionnaire as well as the Long Bowel Disease questionnaire (BDQ). Mayo Clinic Institutional Review Board approved human studies and all participants provided a written informed consent (IRB protocol: 12-006529; ClinicalTrials.gov identifier: NCT03266068).

Treatment ID:

TR002190

Treatment Summary:

A total of 52 PI-IBS patients defined by Rome III criteria and 38 healthy volunteers were recruited. Those with a history of abdominal surgery (except hernia, C-section, hysterectomy, appendectomy or cholecystectomy), inflammatory bowel disease, microscopic colitis, or celiac disease were excluded. Additionally, recruited volunteers were not pregnant at the time of the study. Use of tobacco or alcohol for the duration of the study was prohibited. Following medications were prohibited 7 days prior to study participation: those affecting gastrointestinal transit, serotonergic agents, anti-cholinergic agents, antimuscarinics, narcotics, peppermint oil, antibiotics or new probiotics. Ingestion of artificial sweeteners such as SplendaTM (sucralose), Nutrasweet TM (aspartame), lactulose or mannitol was prohibited for 2 days before the start and during the study. All subjects taking part in the study were asked to complete the Hospital Anxiety and Depression Scale (HADS) and a 7-day bowel diary. All participants completed the Hospital anxiety and depression scale (HADS). PI-IBS patients also completed the Symptom Checklist-90 (SCL-90), IBS Symptom severity scale (IBS-SSS), IBS-quality of life (IBS-QoL) questionnaire as well as the Long Bowel Disease questionnaire (BDQ). Mayo Clinic Institutional Review Board approved human studies and all participants provided a written informed consent (IRB protocol: 12-006529; ClinicalTrials.gov identifier: NCT03266068).

Sample Preparation:

Sampleprep ID:	SP002184
Sampleprep Summary:	Fecal samples were deproteinized with six times volume of cold acetonitrile:methanol (1:1 ratio), kept on ice with intermittent vortexing for 30 minutes at 4C, then centrifuged at 18000xg. 13C6-phenylalanine (3 µl at 250ng/µl) was added as internal standard to each sample prior to deproteinization. The supernatants were divided into 2 aliquots and dried down for analysis on a Quadrupole Time-of-Flight Mass Spectrometer (Agilent Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data were acquired under both positive and negative electrospray ionization conditions over a mass range of 100 - 1200 m/z at a resolution of 10,000-35,000 (separate runs). Metabolite separation was achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters). For each column, the run time is 20 min using a flow rate of 400 ul/min. A total of four runs per sample will be performed to give maximum coverage of metabolites. Samples were injected in duplicate or triplicate, and a quality control sample, made up of a subset of samples from the study was injected several times during a run. All raw data files obtained were converted to compound exchange file format using Masshunter DA reprocessor software (Agilent). Mass Profiler Professional (Agilent) was used for data alignment and to convert each metabolite feature (m/z x intensity x time) into a matrix of detected peaks for compound identification.

Sampleprep ID:

SP002184

Sampleprep Summary:

Fecal samples were deproteinized with six times volume of cold acetonitrile:methanol (1:1 ratio), kept on ice with intermittent vortexing for 30 minutes at 4C, then centrifuged at 18000xg. 13C6-phenylalanine (3 µl at 250ng/µl) was added as internal standard to each sample prior to deproteinization. The supernatants were divided into 2 aliquots and dried down for analysis on a Quadrupole Time-of-Flight Mass Spectrometer (Agilent Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data were acquired under both positive and negative electrospray ionization conditions over a mass range of 100 - 1200 m/z at a resolution of 10,000-35,000 (separate runs). Metabolite separation was achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters). For each column, the run time is 20 min using a flow rate of 400 ul/min. A total of four runs per sample will be performed to give maximum coverage of metabolites. Samples were injected in duplicate or triplicate, and a quality control sample, made up of a subset of samples from the study was injected several times during a run. All raw data files obtained were converted to compound exchange file format using Masshunter DA reprocessor software (Agilent). Mass Profiler Professional (Agilent) was used for data alignment and to convert each metabolite feature (m/z x intensity x time) into a matrix of detected peaks for compound identification.

Combined analysis:

Analysis ID	AN003420	AN003421	AN003422
Analysis type	MS	MS	MS
Chromatography type	Reversed phase	HILIC	HILIC
Chromatography system	Agilent 1290 Infinity	Agilent 1290 Infinity	Agilent 1290 Infinity
Column	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)	Waters Acquity BEH HILIC (150 x 2.1mm,1.7um)	Waters Acquity BEH HILIC (150 x 2.1mm,1.7um)
MS Type	ESI	ESI	ESI
MS instrument type	QTOF	QTOF	QTOF
MS instrument name	Agilent 6550 QTOF	Agilent 6550 QTOF	Agilent 6550 QTOF
Ion Mode	POSITIVE	POSITIVE	NEGATIVE
Units	raw intensity	raw intensity	raw intensity

Chromatography:

Chromatography ID:	CH002530
Chromatography Summary:	Reversed phase
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
Chromatography Type:	Reversed phase

Chromatography ID:	CH002531
Chromatography Summary:	HILIC
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity BEH HILIC (150 x 2.1mm,1.7um)
Chromatography Type:	HILIC

Chromatography ID:	CH002532
Chromatography Summary:	HILIC
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity BEH HILIC (150 x 2.1mm,1.7um)
Chromatography Type:	HILIC

MS:

MS ID:	MS003185
Analysis ID:	AN003420
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	None
Ion Mode:	POSITIVE

MS ID:	MS003186
Analysis ID:	AN003421
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	None
Ion Mode:	POSITIVE

MS ID:	MS003187
Analysis ID:	AN003422
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	None
Ion Mode:	NEGATIVE