DOI: 10.25345/c5pv6bj2w
title: MassIVE MSV000093572 - Quantitative proteomics of Axon Regeneration in Xenopus laevis: A closer look at the Tectum, Retina, Chiasm, and Optic Nerve.
title: In this labeled quantitative proteomics dataset, we profile the proteomic changes in the tectum, retina, chiasm, and optic nerve in transgenic lines of 1 year old Xenopus laevis Tg(islet2bgfp). The frogs had monocular surgery of either a left optic crush injury (crush) or sham surgery (sham) and the matching controls of uninjured optic nerves were collected as a control factor. The Tg(islet2bgfp) frogs were allowed to recover for 12 and 27 days post optic nerve crush before euthanasia and tissue collection. Protein extraction was carried by homogenization of the tissue in extraction buffer (TEAB, NaCl, SDS) via Precellys. During extraction, three internal peptide standards were spiked to measure extraction efficiency. Protein amounts were estimated and normalized across experimental samples. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. TMT (Tandem Mass Tag) labelling was carried out using 6 sets of 17 tags from a 18plex TMT kit for quantification. The samples were fractionated into 9 fractions using Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific). After fractionation and drying of all peptide samples, each TMT sample was spiked with two additional isobarically labelled peptides to be used as an ionization control and cross-sample quantification standard. The combination internal peptides were used to compare and normalize against a future axon regeneration cohort. 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. After animals were euthanized, optic nerves were collected by dissection. There were six experimental conditions and six biological replicates per tissue for a total of 102 samples. Protein extraction was carried out by homogenizing the optic nerve tissue in TEAB, NaCl and SDS. Three synthetic peptide standards 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 6 sets of 17 tags from a 18plex TMT (Tandem Mass Tag) kit for quantification. After combination and drying of all peptide samples, each combined TMT sample was spiked with two additional peptide standards containing isobaric labels. The final concentration of the post extraction peptides was 54uM per plex or 324uM for all six plex's. These standards were spiked in 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. Raw mass spectrometry data files were analyzed using Proteome Discoverer 3.0. The frog 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.