Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA128470

Local Sample ID:	S10
Subject ID:	SU001599
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6
Age Or Age Range:	8 weeks
Gender:	Male and female
Animal Animal Supplier:	Jackson Labs (Bar Harbor, ME USA)

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Subject:

Subject ID:	SU001599
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6
Age Or Age Range:	8 weeks
Gender:	Male and female
Animal Animal Supplier:	Jackson Labs (Bar Harbor, ME USA)

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
S10	SA128470	FL016035	LFD	Diet
S10	SA128470	FL016035	PFOS	Treatment

Collection:

Collection ID:	CO001594
Collection Summary:	The study investigated diet-PFAS interactions and the impact of perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic (PFHxS) on the hepatic proteome and blood lipidomic profiles. The results supported the hypothesis that PFOS and PFHxS increase the risk of metabolic and inflammatory disease induced by diet.
Sample Type:	Blood (whole)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001614
Treatment Summary:	The mice were fed either a 10.5% kcal, low fat diet (LFD) (D12328, Research Diets, New Brunswick), or a 58% kcal, high fat diet (HFD) (D12331, Research Diets, New Brunswick). The mice were assigned to either diet alone, as controls, or to diet containing 0.0003% PFOS or 0.0003% PFHxS. The resulting treatment groups were as follows: low fat diet (LFD), high fat high carbohydrate diet (HFHC), LFD + PFOS (LPFNA), HFHC + PFOS (HPFOS), LFD + PFHxS (LPFHxS), and HFHC + PFHxS (HPFHxS) at n = 6 per treatment group.

Treatment ID:

TR001614

Treatment Summary:

The mice were fed either a 10.5% kcal, low fat diet (LFD) (D12328, Research Diets, New Brunswick), or a 58% kcal, high fat diet (HFD) (D12331, Research Diets, New Brunswick). The mice were assigned to either diet alone, as controls, or to diet containing 0.0003% PFOS or 0.0003% PFHxS. The resulting treatment groups were as follows: low fat diet (LFD), high fat high carbohydrate diet (HFHC), LFD + PFOS (LPFNA), HFHC + PFOS (HPFOS), LFD + PFHxS (LPFHxS), and HFHC + PFHxS (HPFHxS) at n = 6 per treatment group.

Sample Preparation:

Sampleprep ID:	SP001607
Sampleprep Summary:	Blood lipids were isolated for lipidomic analysis according to the Bligh and Dyer method (Bligh and Dyer 1959). The lipidomics was performed at the University of Georgia (Athens, GA). Briefly, blood samples designated for lipidomics were suspended in 1.25 ml of methanol and 1.25 ml of chloroform. Tubes were vortexed for 30 s, allowed to sit for 10 min on ice, centrifuged (300 x g; 5 min), and the bottom chloroform layer was transferred to a new test tube. The extraction steps were repeated three times and the chloroform layer combined. A commercial mix of SPLASH Lipidomix internal standards (Avanti Polar Lipids, Inc.) were spiked into each sample. SPLASH Lipidomix Mass Spec standards includes all major lipid classes at ratios similar to that found in human plasma. The collected chloroform layers were dried under nitrogen, reconstituted with 50 µl of methanol: chloroform (3:1 v/v), and stored at 80ºC until analysis. Lipid content was quantified by determining the level of inorganic phosphorus using the Bartlett Assay (Bartlett 1959).

Sampleprep ID:

SP001607

Sampleprep Summary:

Blood lipids were isolated for lipidomic analysis according to the Bligh and Dyer method (Bligh and Dyer 1959). The lipidomics was performed at the University of Georgia (Athens, GA). Briefly, blood samples designated for lipidomics were suspended in 1.25 ml of methanol and 1.25 ml of chloroform. Tubes were vortexed for 30 s, allowed to sit for 10 min on ice, centrifuged (300 x g; 5 min), and the bottom chloroform layer was transferred to a new test tube. The extraction steps were repeated three times and the chloroform layer combined. A commercial mix of SPLASH Lipidomix internal standards (Avanti Polar Lipids, Inc.) were spiked into each sample. SPLASH Lipidomix Mass Spec standards includes all major lipid classes at ratios similar to that found in human plasma. The collected chloroform layers were dried under nitrogen, reconstituted with 50 µl of methanol: chloroform (3:1 v/v), and stored at 80ºC until analysis. Lipid content was quantified by determining the level of inorganic phosphorus using the Bartlett Assay (Bartlett 1959).

Combined analysis:

Analysis ID	AN002546
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo-Fisher LTQ Orbitrap Elite
Column	Bruker Micron Magic nanoC18 (130mm X 100um,5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Orbitrap Elite Hybrid Ion Trap-Orbitrap
Ion Mode	POSITIVE
Units	Normalized Peak Height

Chromatography:

Chromatography ID:	CH001864
Chromatography Comments:	nanoC18 column (length, 130 mm; i.d., 100 μm; particle size, 5 μm; pore size, 150 Å; max flow rate, 500 nL/min; packing material, Bruker Micron Magic 18)
Instrument Name:	Thermo-Fisher LTQ Orbitrap Elite
Column Name:	Bruker Micron Magic nanoC18 (130mm X 100um,5um)
Flow Rate:	450-500 nL/min
Injection Temperature:	7 °C
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Time Program:	60 mins
Target Sample Temperature:	7 °C
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002364
Analysis ID:	AN002546
Instrument Name:	Thermo Orbitrap Elite Hybrid Ion Trap-Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Lipid structures were identified based on the retention time and subsequent MS/MS spectra. Essentially, we determined structural information through LC-MS/MS and normalization of available lipid standards using LipidMatch. First, lipidomics data processed lipid features using MZmine as described in (Koelmel et al. 2017). Features observed in the blanks were removed using the blank feature filtration method (Patterson et al. 2017). The blank feature filtration method compared to various other filtering methods has been shown to increase the removal of true negatives while decreasing the removal of true positives (Patterson et al. 2017). The resulting MZmine features were annotated using LipidMatch (Koelmel et al. 2017). These annotations are putative, as annotations are based on in-silico MS/MS spectral libraries without matching internal standards for validation and without confirmation using orthogonal approaches (Sumner et al. 2007). The lipid match program then provided a single point calibration using exogenous lipid internal calibrant that best represents the lipid feature (based on lipid class, adduct and retention time). An R script was applied that combined multiple lipid features (adducts) into one feature 4 representing a unique lipid molecule. All open source lipidomics tools are published and available at http://secim.ufl.edu/ secim-tools/.
Ion Mode:	POSITIVE