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MB Sample ID: SA052824

Local Sample ID:27
Subject ID:SU000936
Subject Type:Human serum
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Human

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Subject:

Subject ID:SU000936
Subject Type:Human serum
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Human

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
27SA052824FL010003ControlType

Collection:

Collection ID:CO000930
Collection Summary:Patients with confirmed UC (n = 20) were recruited from either the outpatient endoscopy unit or IBD clinic at Vanderbilt University Medical Center. Patients with confirmed CD (n = 20) were recruited from the IBD clinic at Vanderbilt University Medical Center. IBD diagnosis was determined by accepted clinical and histologic criteria by an IBD specialist. Non-IBD control subjects (n = 20) were recruited from patients undergoing colonoscopy for colorectal cancer screening or other non-IBD related indications. Individuals who were pregnant, had known coagulopathy or bleeding disorders, known renal or hepatic impairment, prior organ transplant, or were unable to give informed consent were excluded. Blood was collected in a vacutainer without additives and allowed to clot. The sample was centrifuged within an hour of collection and serum was aliquoted. Serum samples were snap frozen with dry ice and then stored at –80°C.
Sample Type:Blood

Treatment:

Treatment ID:TR000950
Treatment Summary:Individuals were categorized as either having Crohn disease, Ulcerative colitis, or as non-IBD controls by accepted clinical and histologic criteria by an IBD specialist.

Sample Preparation:

Sampleprep ID:SP000943
Sampleprep Summary:: All samples were maintained at -80oC until processed. Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis.
Sampleprep Protocol Filename:SupplementalMaterial1.docx

Combined analysis:

Analysis ID AN001462 AN001463 AN001464 AN001465
Analysis type MS MS MS MS
Chromatography type Reversed phase Reversed phase Reversed phase HILIC
Chromatography system Waters Acquity Waters Acquity Waters Acquity Waters Acquity
Column Waters Acquity BEH C18 (100 x 2.1mm,1.7um) Waters Acquity BEH C18 (100 x 2.1mm,1.7um) Waters Acquity BEH C18 (100 x 2.1mm,1.7um) Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
MS Type ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE POSITIVE NEGATIVE NEGATIVE
Units raw area counts raw area counts raw area counts raw area counts

Chromatography:

Chromatography ID:CH001028
Chromatography Summary:One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient-eluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). A 2nd aliquot was also analyzed using acidic positive ion conditions, however it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the same C18 column using methanol, acetonitrile, water, 0.05% PFPA, and 0.01% FA and was operated at an overall higher organic content. A 3rd aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however with 6.5mM ammonium bicarbonate at pH 8.
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (100 x 2.1mm,1.7um)
Chromatography Type:Reversed phase
  
Chromatography ID:CH001029
Chromatography Summary:The 4th aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM ammonium formate, pH 10.8.
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Solvent A:100% water; 10 mM ammonium formate, pH 10.8
Solvent B:100% acetonitrile; 10 mM ammonium formate, pH 10.8
Chromatography Type:HILIC

MS:

MS ID:MS001350
Analysis ID:AN001462
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Waters ACQUITY ultra-performance liquid chromatography (UPLC) Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution
Ion Mode:POSITIVE
Resolution Setting:35,000
  
MS ID:MS001351
Analysis ID:AN001463
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Waters ACQUITY ultra-performance liquid chromatography (UPLC) Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution
Ion Mode:POSITIVE
Resolution Setting:35,000
  
MS ID:MS001352
Analysis ID:AN001464
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Waters ACQUITY ultra-performance liquid chromatography (UPLC) Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution
Ion Mode:NEGATIVE
Resolution Setting:35,000
  
MS ID:MS001353
Analysis ID:AN001465
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Waters ACQUITY ultra-performance liquid chromatography (UPLC) Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution
Ion Mode:NEGATIVE
Resolution Setting:35,000
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