MassIVE MSV000093905

Complete Public PXD048812

Quantitative Proteomics of axon regeneration promoted by Gangliosides and MTOR-Activating Small Molecules

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Description

The ability to regenerate damaged central nervous system (CNS) axons of existing neurons is a significant challenge in treating conditions of neurodegenerative disorders such as glaucoma. To achieve neuroregeneration, we evaluated small molecules to target the MTOR pathway for axonal growth. In this study, 3-month-old C57BL/6J mice with equal gender distribution, a total of 2 lipid gangliosides and 4 small molecules were used. These are vehicle only, GM1, GM3, V0-Ophic, 3BDO, and Rapamycin. For each mouse, optic nerve crush was performed in one eye. At 2 days post injury, the same eye had an intravitreal injection of one or a combination of the small molecules noted above. Two days before euthanasia (Day 12), fluorophore 488 coupled cholera toxin B (CTB-488) was injected intravitreally for axonal imaging at endpoints. After animals were euthanized, optic nerves were collected by dissection. Three identical biological replicates for each treatment group (that were not injected with CTB-488) were analyzed by mass spectrometry. Protein extraction was carried out by homogenization of the tissue in extraction buffer (Triethylammonium bicarbonate buffer, Sodium Chloride and Sodium dodecyl sulfate). Protein amounts were estimated and normalized across experimental samples via densitometry. Samples were reduced using Tris(2-carboxyethyl) phosphine hydrochloride, alkylated with iodoacetamide, and digested overnight with trypsin. TMT (Tandem Mass Tag) labelling was carried out using 3 sets of 13 tags from a 16plex TMT kit for quantification. Samples were combined, dried, and fractionated. Untargeted liquid chromatography-mass spectrometry was performed on an Easy-nLC 1000 liquid chromatograph coupled to a QExactive mass spectrometer (LC-MS/MS). Data analysis was performed using Proteome Discoverer 3.0 and Graph Pad Prism 10. Raw mass spectrometry data files were analyzed using Proteome Discoverer 3.0. The mouse 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. [doi:10.25345/C5JH3DD23] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Axon Regeneration, Quantitative Proteomics, TMT labeled, Mouse, Optic Nerve

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Principal Investigators:
(in alphabetical order) 		Sanjoy Bhattacharya, University of Miami, N/A
Submitting User: sbhattacharya
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