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Lipidomics dataset of PTEN deletion-induced nerve regeneration mouse model

ABSTRACT: We present the lipidome of adult PTENloxP/loxP mice subjected to intravitreal injection of adeno-associated viruses expressing Cre (AAV-Cre) as a model of regeneration. At 4-week-old, PTENloxP/loxP mice were intravitreally-injected with 2-3 μl of either AAV-Cre (KO) or AAV-PLAP (control), and two weeks later optic nerve crush was performed. At indicated time-points after crush (0 days, 7 days, 14 days), mice were euthanized and optic nerves were immediately dissected out, and then flash frozen on dry ice. The Bligh and Dyer method was used for lipid extraction, followed by mass spectrometry lipid profiling with a Q-Exactive Orbitrap Liquid Chromatography-Mass Spectrometer (LC MS-MS). The raw scans were analysed with LipidSearch 4.2 and the statistical analysis was conducted through Metaboanalyst 4.0

ORGANISM(S): Mouse Mus Musculus

TISSUE(S): Eye Tissue

DISEASE(S): Eye Disease

SUBMITTER: Sanjoy Bhattacharya

PROVIDER: ST001477 | MetabolomicsWorkbench | Mon Aug 31 00:00:00 BST 2020

REPOSITORIES: MetabolomicsWorkbench

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Project description:Zebrafish (Danio Rerio) have the capacity for successful adult optic nerve regeneration, unlike mammals. Optic nerve regeneration is frequently studied using optic nerve crush (ONC). For ONC, animals were deeply anesthetized in 0.033% tricaine methane-sulfonate (MS-222). The right optic nerve was exposed by gently removing the connective tissue on the dorsal half of the eye and rotating the eye ventrally out of the orbit with a pair of number 5 forceps. A nerve crush was then performed using number 5 forceps to crush the nerve ~0.5 to 1 mm from the optic nerve head for 5 seconds. Success of crush was assessed by identifying the generation of a translucent stripe in the nerve that completely separated two areas of white myelination with no bleeding. Fish were then revived in fresh aquatic system water in individual tanks. After 1 hour the tanks were returned to the fish system and animals were maintained normally with daily feeding until 3 days post injury. Female and male (6 month to 1 year old) Zebrafish optic nerves (right side/OD) were crushed and collected three days after. The associated retinas and tecta were also collected under the same conditions. Contralateral, uninjured optic nerves, retinas and tecta were collected as controls. For tissue collection, animals were euthanized by overdose of MS-222 (>400mg/L) followed by dissection. The tissue was collected from the optic nerve head to the optic chiasm. The tissues were immediately frozen on dry ice. Optic nerve samples were pooled for each category (female crush, female control, male crush, male control) and pooled at n = 31 to obtain sufficient protein concentrations for analysis (pooled optic nerves served as one sample). Retina and tectum samples were pooled using the same categories (female crush, female control, male crush, male control) at n = 10-12 (pooled tissue served as one sample). Protein extraction was carried out by homogenizing the optic nerve tissue in TEAB, NaCl and SDS. Three synthetic peptide standards (Regen III) were added to the samples during the extraction to measure extraction efficiency. Each standard was spiked into samples for a total concentration of 48uM per standard. After extraction was carried out, protein amounts were estimated using dot blot densitometry and ImageJ and normalized to 50ug/ul. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. All samples were labelled using 2 sets of 14 tags from a 18plex TMT (Tandem Mass Tag) kit (A52045: Thermo Fisher Scientific, Waltham, MA) for quantification. After combination and drying of all peptide samples, the samples were fractionated into 9 fractions using Pierce High pH Reversed-Phase Peptide Fractionation Kit (84868: Thermo Fisher Scientific, Waltham, MA). After fractionation and drying of all peptide samples, each fractionated TMT sample was spiked with two additional peptide standards (Regen II) containing isobaric labels. The final concentration of the post extraction spiked peptides was 54uM per plex. These standards (Regen II) were spiked directly before mass spectrometry analysis to be used as an ionization control. Additionally, all five standards serve as a normalization method that may be used to compare protein abundance data across multiple datasets. Untargeted liquid chromatography-mass spectrometry was performed on an Easy-nLC 1000 liquid chromatograph coupled to a QExactive mass spectrometer (LC-MS/MS). Raw mass spectrometry data files were analyzed using Proteome Discoverer 3.0. The Danio rerio proteome was downloaded from UniProt and used as the target database for protein identification. Max missed cleavage site was set to 2 and minimum peptide length to 6 . Precursor Mass Tolerance was set to 10ppm and Fragment Mass Tolerance to 0.02 Da. Post-translational modifications for experimental proteins included oxidation, acetylation, carbamidomethylation and TMTpro. The Normalization was set to total peptide amount and confidence to low. Two additional local databases were created for the pre- and post- extraction peptides and ran separately from experimental protein identification.

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Lipidomics dataset of PTEN deletion-induced nerve regeneration mouse model

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