DOI: 10.25345/c5ks6jg78
title: MassIVE MSV000096167 - Quantitative Proteomics of optic nerve axon regeneration in C57BL/6J Mus musculus with HDAC3 gene knockout utilizing Regen V Standardization.
title: The optic nerve crush (ONC) in mouse is a commonly utilized model for investigation of axon regeneration, which has been utilized in a mouse model with the HDAC3 gene knocked out and a wildtype floxed mouse control (both were subjected to ONC). Floxed C57BL/6J mice were developed with LoxP sites on either side of exon 7 of HDAC3. The HDAC3 KO specific for macrophage and microglial cells generated for the experimental condition was created by crossing LysM Cre mice with floxed mice. Histone deacetylase 3 (HDAC3) is an enzyme that acts by deacetylating lysine residues of histones. HDAC3 causes the DNA to separate from the histone due to their deacetylation, allowing it to be accessible for gene expression that in turn positively impact many processes including axon regeneration. ONC was performed on 10 to 12 week old male mice under anesthesia. The surrounding structures of the left eye were dissected to expose the optic nerve and it was crushed 1 to 2mm from the globe. The mice were euthanized, and the optic nerves of both mice groups were harvested at several time points post ONC: 0 days, 7 days, and 14 days. Once collected, the optic nerves were minced via homogenization in Precellys tubes and with an added buffer made of sodium chloride (NaCl), Triethylammonium bicarbonate (TEAB), and Sodium dodecyl sulfate (SDS). To analyze the efficiency of the process, Regen III peptides were also added to the buffer solution in each sample that served to inform protein extraction efficiency. To normalize the protein amount in the samples, dot blot densitometry was performed with a bovine serum albumin (BSA) standard and results were evaluated with ImageJ software. Protein samples were then subjected to a digestion process that included a TEAB dissolution buffer with an amount determined by densitometry specific to each sample, SDS, and reducing agent tris (2 carboxyethyl) phosphine (TCEP). After adding TCEP to the samples and incubating them at 60C, the samples were alkylated with iodoacetamide. After another incubation at room temperature and in the dark, Trypsin was added several times to each sample and then the samples digested overnight. After trypsin digestion, a detergent removal was performed, and the samples were cleaned with C18 zip tips. The quantification process was started with one group of 18 tags from an 18plex Tandem Mass Tag (TMT) kit for TMT labelling of the 18 samples. After being labeled, the samples were incubated at room temperature for an hour, then 5% hydroxylamine was added to each sample tube to stop the reaction. An equal amount from each sample tube was combined into a new microcentrifuge tube and then was dried. After drying, the sample was reconstituted in 47uL of 2% ACN with water and 0.1% Formic Acid, 1uL Trypsin Digested BSA, and 2 uL Regen II peptide mixture. It was sonicated in an ultrasonic bath for 20 minutes for total solubilization. The sample was then transferred to the respective HPLC vial. Proteins were separated using a Thermo Scientific Easy nLC 1000 system and 1 uL was injected onto an Acclaim PepMap RSLC column (75 um x 15 cm, nanoViper C18, 2 um, 100A, SN: 20635405). The flow rate was 300 nL/min. Mobile phase A consisted of water with 0.1% formic acid (v/v) and mobile phase B was acetonitrile with 0.1% formic acid (v/v). Samples were ionized and detected on a NanoSpray Flex ion source coupled to a Q Exactive mass spectrometer (Thermo Scientific). Spray voltage was set to 2.42 kV and capillary temperature to 300C. S Lens RF Level was set to 55.0. For full scan, mass range was 375 to 1400 m/z, resolution was 70,000, and microscans was 1. The AGC target was 3e6 and maximum injection time was 50 ms. For ddMS2, mass range was 200 to 2000 m/z, resolution was 35,000, AGC target was 1e5, and maximum injection time was 100 ms. The instrument was set to Top 10, isolation window 1.2 m/z and the NCE to 32. The intensity threshold was 2.04e4 and dynamic exclusion was 30.0 s. Raw mass spectrometry files were processed on Proteome Discoverer 3.0. The mouse proteome was downloaded from UniProt and used as the target database for protein identification. Two additional local databases were created. One containing the Regen V internal standard peptide sequences, and the other containing BSA peptide sequences. The identification of the Regen V and BSA standards were ran in separate Proteome Discoverer workflows to identify the internal standards separately from experimental proteins. 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. Post translational modifications of the Regen II, (biotin, carbon 13, and nitrogen 15) were added to ensure correct identification of the modified standards. The Normalization was set to total peptide amount and confidence to low.