Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA245374

Local Sample ID:	B648_Hippocampus
Subject ID:	SU002542
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	APOE-targeted replacement mice homozygous for human E3 (B6.129P2-Apoe^tm2(APOE3)Mae N8, Taconic #1548-F) or human E4 (B6.129P2- Apoe^tm3(APOE4)Mae N8, Taconic #1549-F) alleles and crossed to the 5x Familial Alzheimer's Disease (5XFAD) model (MMRRC #34840, B6SJL^Tg( APPSwFlLon,PSEN1M146LL286V)6799Vas/Mmjax))
Age Or Age Range:	3 months, 12 months, 24 months
Gender:	Female

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Subject:

Subject ID:	SU002542
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	APOE-targeted replacement mice homozygous for human E3 (B6.129P2-Apoe^tm2(APOE3)Mae N8, Taconic #1548-F) or human E4 (B6.129P2- Apoe^tm3(APOE4)Mae N8, Taconic #1549-F) alleles and crossed to the 5x Familial Alzheimer's Disease (5XFAD) model (MMRRC #34840, B6SJL^Tg( APPSwFlLon,PSEN1M146LL286V)6799Vas/Mmjax))
Age Or Age Range:	3 months, 12 months, 24 months
Gender:	Female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
B648_Hippocampus	SA245374	FL030742	Hippocampus	Brain Region
B648_Hippocampus	SA245374	FL030742	E3_young	Group
B648_Hippocampus	SA245374	FL030742	E3/E3	APOE genotype
B648_Hippocampus	SA245374	FL030742	Wild-type	5XFAD Genotype
B648_Hippocampus	SA245374	FL030742	3 months	Age

Collection:

Collection ID:	CO002535
Collection Summary:	Three different mice for each experimental group (18 mice in total) were anesthetized via 5.0% isoflurane before exsanguination and transcardial perfusion with ice-cold Dulbecco’s phosphate buffered saline (DPBS; Gibco # 14040133). Following perfusion, hemibrains were quickly removed, leaving behind brainstem and cerebellum. Hemibrains were immediately placed in OCT compound (Fisher HealthCare Tissue Plus O.C.T. Compound Clear 4585) and gently lowered into isopentane (Sigma-Aldrich 2- Methylbutane M32631) in a beaker surrounded by dry ice (isopentane chilled to approximately -70°C). Hemibrains were submerged for 60 seconds, placed on dry ice, wrapped in aluminum foil, and stored at -80°C until sectioning.
Sample Type:	Brain

Treatment:

Treatment ID:	TR002554
Treatment Summary:	All mice were group housed in sterile micro-isolator cages (Lab Products, Maywood, NJ), and fed autoclaved food and acidified water ad libitum. No additional treatment was applied, with the only comparisons being across different genotypes and ages as specified in the study design table.

Sample Preparation:

Sampleprep ID:	SP002548
Sampleprep Summary:	Prepared brain hemispheres were cryosectioned to 10 μm thick coronal sections at approximately Bregma -2.00 mm. Brain sections were mounted on glass slides and prepared for MALDI MSI as follows: After desiccation for one hour, slides were sprayed with 14 passes of 7mg/mL N-(1-Naphthyl) ethylenediamine dihydrochloride (NEDC) matrix (Sigma) in 70% methanol (HPLC-grade, Sigma) which was applied at 0.06mL/min with a 3mm offset and a velocity of 1200mm/min at 30°C and 10psi using the M5 Sprayer with a heated tray of 50°C. Slides were used immediately or stored in a desiccator until analysis.

Sampleprep ID:

SP002548

Sampleprep Summary:

Prepared brain hemispheres were cryosectioned to 10 μm thick coronal sections at approximately Bregma -2.00 mm. Brain sections were mounted on glass slides and prepared for MALDI MSI as follows: After desiccation for one hour, slides were sprayed with 14 passes of 7mg/mL N-(1-Naphthyl) ethylenediamine dihydrochloride (NEDC) matrix (Sigma) in 70% methanol (HPLC-grade, Sigma) which was applied at 0.06mL/min with a 3mm offset and a velocity of 1200mm/min at 30°C and 10psi using the M5 Sprayer with a heated tray of 50°C. Slides were used immediately or stored in a desiccator until analysis.

Combined analysis:

Analysis ID	AN004002
Analysis type	MS
Chromatography type	None (Direct infusion)
Chromatography system	none
Column	none
MS Type	MALDI
MS instrument type	QTOF
MS instrument name	Waters Synapt G2 XS QTOF
Ion Mode	NEGATIVE
Units	Intensity per pixel

Chromatography:

Chromatography ID:	CH002955
Chromatography Summary:	Not applicable (MALDI MSI)
Instrument Name:	none
Column Name:	none
Column Temperature:	N/A
Flow Gradient:	N/A
Flow Rate:	N/A
Solvent A:	N/A
Solvent B:	N/A
Chromatography Type:	None (Direct infusion)

MS:

MS ID:	MS003750
Analysis ID:	AN004002
Instrument Name:	Waters Synapt G2 XS QTOF
Instrument Type:	QTOF
MS Type:	MALDI
MS Comments:	For the detection of lipids, a Waters SynaptG2-Xs high-definition mass spectrometer equipped with traveling wave ion mobility was employed with the following parameters. The laser was operating at 2000 Hz with an energy of 300 AU and spot size of 50 μm at X and Y coordinates of 100μm with mass range set at 50 – 1000 m/z in negative mode. MALDI-MSI data files were processed to adjust for mass drift during the MALDI scan and to enhance image quality and improve signal-tonoise ratio using an algorithm available within the High-Definition Imaging (HDI) software (Waters Corp). To adjust for mass drift during the MALDI scan, raw files were processed using a carefully curated list of 20 MALDI NEDC matrix peaks (m/z), 26 small molecule MALDI peaks(m/z), and 24 lipid peaks(m/z). Files were processed at a sample duration of 10 sec at a frequency rate of 0.5 min, and an m/z window of 0.1 Da, using an internal lock mass of previously defined metabolite of taurine 124.007 m/z with a tolerance of 1amu and a minimum signal intensity of 100,000 counts. Data acquisition spectrums were uploaded to the HDI software for the generation of lipid images. Regions of interest (ROIs) were user defined by a blinded investigator using anatomical reference points based on the mouse Allen Brain atlas. For all pixels defined within a ROI, peak intensities were averaged and normalized by total ion current (TIC) and number of pixels.
Ion Mode:	NEGATIVE