DOI: 10.25345/c59z90p3s
title: MassIVE MSV000093512 - Quantitative proteomics with Regen V standardization of PTEN deletion-induced optic nerve regeneration mouse models and controls.
title: A quantitative proteomic profiling was performed on PTEN deletion-induced optic nerve regeneration mouse models and controls. At postnatal day 28 (week 4), PTENloxP/loxP mice were subjected to intravitreal injection (2-3ul) of adeno-associated viruses expressing Cre (AAV2-Cre) or control placenta alkaline phosphatase (AAV2-PLAP). Optic Nerve crush was performed 2 weeks post AAV injections at three times points (0, 7, 14 days post injury) and collected for proteomics analysis. A protein extraction was carried out by homogenization of the tissue in extraction buffer (TEAB, NaCl and SDS). During extraction, three internal peptide standards, named as Regen III, 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 1 sets of 18 tags from a 18plex TMT kit for quantification. After combination and drying of all peptide samples, each TMT sample was spiked with two additional isobarically labelled human peptides to be used as an ionization control and cross-sample quantification standard (Regen II). Overall, Regen V standards (refers to a combination of Regen III and Regen II, for extraction and ionization normalization) were used to compare and normalize against any future axon regeneration sample 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 6 experimental conditions and three biological replicates for each condition for a total of 18 nerves. Protein extraction was carried out by homogenizing the optic nerve tissue in TEAB, NaCl and SDS. Three synthetic bacterial 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 18uM 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 1 set of 18 tags from a 18plex TMT (Tandem Mass Tag) kit for quantification. After combination and drying of all peptide samples, the samples were fractionated via Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher). Each combined TMT sample was spiked with two additional human peptide standards containing isobaric labels (Regen II). The final concentration of Regen II was 54uM. These standards were spiked in directly before mass spectrometry analysis to be used as an ionization control. Additionally, all five standards, known as Regen V (Regen III + Regen II), 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 mouse proteome was downloaded from UniProt and used as the target database for protein identification. Two additional local databases were created. One contains the Regen V internal standard peptide sequences, and the other contains BSA peptide sequences. The identification of the Regen V and BSA standards was run in a separate Proteome Discoverer workflow targeted 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 during this step to ensure correct identification of the modified standards. The Normalization was set to total peptide amount and confidence to low.