Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA016111

Local Sample ID:	10303Y
Subject ID:	SU000383
Subject Type:	Animal
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

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Subject:

Subject ID:	SU000383
Subject Type:	Animal
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
10303Y	SA016111	FL003506	-	Severe_Exacerbations
10303Y	SA016111	FL003506	-	Chronic_Bronchitis
10303Y	SA016111	FL003506	3	GoldStage
10303Y	SA016111	FL003506	1	Gender
10303Y	SA016111	FL003506	-	SmokingStatus

Collection:

Collection ID:	CO000377
Collection Summary:	Subjects had fresh frozen plasma collected using a p100 tube (BD) at two COPDGene sites (National Jewish Health and University of Iowa)
Sample Type:	Blood

Treatment:

Treatment ID:	TR000397
Treatment Summary:	Association analysis was done between human subjects and statistical analysis performed using data collected from subjects using covariates and phenotypes as outcomes.

Sample Preparation:

Sampleprep ID:	SP000390
Sampleprep Summary:	Sphingomyelins, dihydrosphingomyelins, ceramides and dihydroceramides: A modified Bligh-Dyer extraction method was used in the presence of internal standards (IS). Sample analysis was performed in four batches using a Shimadzu 10A HPLC system coupled to a TSQ Quantum Ultra triple quadrupole mass spectrometer operated in SRM mode under ESI(+). A Thermo Betasil C18 column was employed to achieve the optimal sensitivity and separation. Data processing was conducted with Xcalibur software (Thermo) to obtain the peak area ratios of analytes to the corresponding internal standards (Supplemental Table 2). A pooled lipid extract from study samples was used as a quality control (QC) sample, and was injected between every 6 study samples to verify the instrument consistency. The peak area ratios were then normalized to the average of QC peak area ratios in the same batch, so that the results for different batches were brought to the same baseline. Sphingoid bases, ceramide-1-phosphate, monohexosylsphingosine, monohexosylceramides, dihexosylceramides, trihexosylceramides, monohydroxylated monohexosylceramides, monohydroxylated dihexosylceramides, sulfatides, and gangliosides: Plasma protein was precipitated with methanol, followed by supernatant collection, drying, and reconstituting with 1:1 methanol/water in the presence of internal standards. Duplicate sample analysis (1 st analysis and 2 nd analysis) was performed in four batches with an online trapping API-4000 system, (consists of a API-4000 mass spectrometer, a Leap PAL autosampler, an Agilent 1100 HPLC, and a Shimadzu HPLC) operated in multiple reaction monitoring (MRM) mode under ESI(+) for all species except sulfatides and E2gangliosides ESI(-). An Agilent Eclipse XRB-C18 column was used for sphingoid bases and ceramide-1- phosphate and a Varian Metasil C18 column was used for analysis of monohexosylsphingosine, mono-, di-, tri- hexosylceramides as well as their monohydroxylated ceramides. An Agilent Zobax Eclipse Plus C18 column was used for analysis of sulfatides and gangliosides. A Thermo Betasil C18 trapping column was also used for all analytes. Data processing was conducted with Analyst software (Applied Sciences) to obtain the peak area ratios of analytes to the corresponding internal standards. A pooled lipid extract from study samples was used as a quality control (QC) sample, and was injected between every 10 study samples to verify the instrumental consistency. All duplicated sample runs were averaged. The values for each sphingolipid were peak area ratios that were normalized to internal standard and then normalized to QC standards. Values were relative peak area ratios and unitless.

Sampleprep ID:

SP000390

Sampleprep Summary:

Sphingomyelins, dihydrosphingomyelins, ceramides and dihydroceramides: A modified Bligh-Dyer extraction method was used in the presence of internal standards (IS). Sample analysis was performed in four batches using a Shimadzu 10A HPLC system coupled to a TSQ Quantum Ultra triple quadrupole mass spectrometer operated in SRM mode under ESI(+). A Thermo Betasil C18 column was employed to achieve the optimal sensitivity and separation. Data processing was conducted with Xcalibur software (Thermo) to obtain the peak area ratios of analytes to the corresponding internal standards (Supplemental Table 2). A pooled lipid extract from study samples was used as a quality control (QC) sample, and was injected between every 6 study samples to verify the instrument consistency. The peak area ratios were then normalized to the average of QC peak area ratios in the same batch, so that the results for different batches were brought to the same baseline. Sphingoid bases, ceramide-1-phosphate, monohexosylsphingosine, monohexosylceramides, dihexosylceramides, trihexosylceramides, monohydroxylated monohexosylceramides, monohydroxylated dihexosylceramides, sulfatides, and gangliosides: Plasma protein was precipitated with methanol, followed by supernatant collection, drying, and reconstituting with 1:1 methanol/water in the presence of internal standards. Duplicate sample analysis (1 st analysis and 2 nd analysis) was performed in four batches with an online trapping API-4000 system, (consists of a API-4000 mass spectrometer, a Leap PAL autosampler, an Agilent 1100 HPLC, and a Shimadzu HPLC) operated in multiple reaction monitoring (MRM) mode under ESI(+) for all species except sulfatides and E2gangliosides ESI(-). An Agilent Eclipse XRB-C18 column was used for sphingoid bases and ceramide-1- phosphate and a Varian Metasil C18 column was used for analysis of monohexosylsphingosine, mono-, di-, tri- hexosylceramides as well as their monohydroxylated ceramides. An Agilent Zobax Eclipse Plus C18 column was used for analysis of sulfatides and gangliosides. A Thermo Betasil C18 trapping column was also used for all analytes. Data processing was conducted with Analyst software (Applied Sciences) to obtain the peak area ratios of analytes to the corresponding internal standards. A pooled lipid extract from study samples was used as a quality control (QC) sample, and was injected between every 10 study samples to verify the instrumental consistency. All duplicated sample runs were averaged. The values for each sphingolipid were peak area ratios that were normalized to internal standard and then normalized to QC standards. Values were relative peak area ratios and unitless.

Combined analysis:

Analysis ID	AN000594
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Shimadzu 10A
Column	Thermo Betasil C18
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Shimadzu 10A
Ion Mode	POSITIVE
Units	Peak Area Ratio

Chromatography:

Chromatography ID:	CH000425
Chromatography Summary:	A modified Bligh-Dyer extraction method was used in the presence of internal standards (IS). Sample analysis was performed in four batches with a Shimadzu 10A HPLC system coupled to a TSQ Quantum Ultra triple quadrupole mass spectrometer operated in SRM mode under ESI(+). A Thermo Betasil C18 column was employed to achieve the optimal sensitivity and separation.
Instrument Name:	Shimadzu 10A
Column Name:	Thermo Betasil C18
Chromatography Type:	Reversed phase

MS:

MS ID:	MS000530
Analysis ID:	AN000594
Instrument Name:	Shimadzu 10A
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	POSITIVE