Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA236808

Local Sample ID:	120091159
Subject ID:	SU002444
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU002444
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
120091159	SA236808	FL029315	control_mid-childhood	Treatment

Collection:

Collection ID:	CO002437
Collection Summary:	Participants of this study included 122 children from the multi-center NIAID Consortium for Food Allergy Research (CoFAR) Observational Study (CoFAR2) who provided stool samples at both infancy and mid-childhood (these are the factors listed in the table). The recruitment and clinical characteristics of these CoFAR2 subjects have been previously described. Briefly, 511 children were recruited at age 3 to 15 months from five US sites (New York, NY, Baltimore, MD, Little Rock, AR, Denver, CO, Durham, NC).16 The cohort was designed as a longitudinal study of infants at high risk for developing peanut allergy, and inclusion criteria included likely egg allergy, milk allergy, and/or moderate to severe atopic dermatitis with a positive egg and/or milk skin prick test at enrollment, but no known peanut allergy. 16 Clinical phenotyping of the subjects, including assessments of peanut allergy, egg allergy, milk allergy, and atopic dermatitis, were performed at 6-12 month intervals between enrollment at infancy and mid-childhood (mean age 9 years, SD 0.6 years). All subjects provided stool samples at baseline and were invited to submit a follow up sample at mid-childhood. Stool samples were collected from 492 of the children at baseline and from 122 of the children at mid-childhood. Samples were immediately stored at -80 o C upon receipt.
Sample Type:	Feces

Treatment:

Treatment ID:	TR002456
Treatment Summary:	Children were categorized as PA if they developed peanut allergy by mid-childhood and not peanut allergic (NPA) if they did not develop peanut allergy by mid-childhood. In study design table, subjects were sampled in infancy and then again mid childhood and placed into either PA or NPA for the purpose of this study. Peanut allergy was defined based on: (1) confirmed IgE-mediated reaction (e.g. positive doctor supervised oral food challenge to peanut and sensitization to peanut), or (2) convincing IgEmediated reaction (e.g. convincing reaction and sensitization to peanut) at any visit.16 This was “alternate definition 1” of the parent CoFAR2 study16 . We used this definition rather than the main definition to avoid inclusion of less convincing peanut allergy in case ascertainment, as the main definition16 also included those with peanut sensitization but no history of reaction.

Treatment ID:

TR002456

Treatment Summary:

Children were categorized as PA if they developed peanut allergy by mid-childhood and not peanut allergic (NPA) if they did not develop peanut allergy by mid-childhood. In study design table, subjects were sampled in infancy and then again mid childhood and placed into either PA or NPA for the purpose of this study. Peanut allergy was defined based on: (1) confirmed IgE-mediated reaction (e.g. positive doctor supervised oral food challenge to peanut and sensitization to peanut), or (2) convincing IgEmediated reaction (e.g. convincing reaction and sensitization to peanut) at any visit.16 This was “alternate definition 1” of the parent CoFAR2 study16 . We used this definition rather than the main definition to avoid inclusion of less convincing peanut allergy in case ascertainment, as the main definition16 also included those with peanut sensitization but no history of reaction.

Sample Preparation:

Sampleprep ID:	SP002450
Sampleprep Summary:	Stool samples were weighed into bead blaster tubes with zircon beads and homogenized in 100% methanol to a final concentration of 10mg/mL including deuterated SCFA internal standards Chun et al. p.22 (CDN Isotopes). 46 Control samples were created by combining sample homogenates into a 1mL of pooled sample (pooled control) or 1mL of methanol (instrument blank). A portion of the pooled control was then subjected to three successive rounds of extraction to deplete SCFAs but retain the insoluble particles to generate null matrix material. A standard curve for the cocktail of SCFAs was prepared in null matrix, and these samples were extracted side-by-side with the study samples and sequence blanks (either methanol or null matrix). The resulting SCFA extracts were then derivatized as described47 for liquid chromatography tandem mass spectrometry (LCMS) with methanol being used instead of acetonitrile. Samples were analyzed with a QExactive HF coupled to a Dionex Ultimate 3000. A Waters BEH C18 column (2.1 x 150mm, 1.7µm) was used with Buffer A as 0.1% formic acid in water, and Buffer B as 0.1%formic acid in acetonitrile. A gradient elution was used (15%B to 55%B in 9 min, 200µL/min) and the mass spectrometer was operated in negative ion mode (HESI, -3.5kV). The order of acquisition was randomized and several control blocks including blanks and external standards were interspersed throughout the run to assess instrument performance and quality control of the derivatization protocol. All analytes were corrected to their respective internal standard, and the resulting ratio was interpolated against the matrix-controlled standard curve to quantify the level of SCFA in each sample. The limit of detection was determined from null-matrix samples for each analyte, and this limit was imputed for any point falling below the limit of detection. The interpolated values were used for the downstream analyses, and the imputed ratio threshold values were used for samples below the limit of detection.

Sampleprep ID:

SP002450

Sampleprep Summary:

Stool samples were weighed into bead blaster tubes with zircon beads and homogenized in 100% methanol to a final concentration of 10mg/mL including deuterated SCFA internal standards Chun et al. p.22 (CDN Isotopes). 46 Control samples were created by combining sample homogenates into a 1mL of pooled sample (pooled control) or 1mL of methanol (instrument blank). A portion of the pooled control was then subjected to three successive rounds of extraction to deplete SCFAs but retain the insoluble particles to generate null matrix material. A standard curve for the cocktail of SCFAs was prepared in null matrix, and these samples were extracted side-by-side with the study samples and sequence blanks (either methanol or null matrix). The resulting SCFA extracts were then derivatized as described47 for liquid chromatography tandem mass spectrometry (LCMS) with methanol being used instead of acetonitrile. Samples were analyzed with a QExactive HF coupled to a Dionex Ultimate 3000. A Waters BEH C18 column (2.1 x 150mm, 1.7µm) was used with Buffer A as 0.1% formic acid in water, and Buffer B as 0.1%formic acid in acetonitrile. A gradient elution was used (15%B to 55%B in 9 min, 200µL/min) and the mass spectrometer was operated in negative ion mode (HESI, -3.5kV). The order of acquisition was randomized and several control blocks including blanks and external standards were interspersed throughout the run to assess instrument performance and quality control of the derivatization protocol. All analytes were corrected to their respective internal standard, and the resulting ratio was interpolated against the matrix-controlled standard curve to quantify the level of SCFA in each sample. The limit of detection was determined from null-matrix samples for each analyte, and this limit was imputed for any point falling below the limit of detection. The interpolated values were used for the downstream analyses, and the imputed ratio threshold values were used for samples below the limit of detection.

Combined analysis:

Analysis ID	AN003846
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000 RS
Column	Waters Acquity BEH C18 (150 x 2mm,1.7um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	POSITIVE
Units	Absolute Intensity

Chromatography:

Chromatography ID:	CH002848
Chromatography Summary:	Previously derivatized short chain fatty acids were separated using RP column in a targeted method. Intrinsic background was accessed by using derivatized blanks and null matrix blanks. Analytes were detected by direct coupling to MS in a targeted PRM method.
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Waters Acquity BEH C18 (150 x 2mm,1.7um)
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003588
Analysis ID:	AN003846
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	All analytes were corrected to their respective internal standard, and the resulting ratio was interpolated against the matrix-controlled standard curve to quantify the level of SCFA in each sample. The limit of detection was determined from null-matrix samples for each analyte, and this limit was imputed for any point falling below the limit of detection. The interpolated values were used for the downstream analyses, and the imputed ratio threshold values were used for samples below the limit of detection
Ion Mode:	POSITIVE