DOI: 10.25345/c5gx4552z
title: MassIVE MSV000093441 - Quantitative proteomics with Regen V standardization of Optogenetics Induced Axon Regeneration.
title: This labeled quantitative proteomics dataset was collected from a transgenic channel rhodopsin mouse model (Chr2) subjected to light stimulation after traumatic optic nerve crush (ONC) was performed. Mouse models expressing channel rhodopsin were subjected to therapeutic light stimulation promoting axon regeneration of retinal ganglion cell (RGC) axons post optic nerve crush. Experimental mice and wild type control mice were euthanized, and optic nerves were collected. A protein extraction was carried out by careful mincing 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 3 sets of 12 tags from a 16plex 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 12 experimental conditions and three biological replicates for each condition for a total of 36 nerves. Protein extraction was carried out by mincing 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 36uM per standard. After extraction was carried out, protein amounts were estimated using dot blot densitometry and ImageJ and normalized to 70ug/ul. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. Prior to labelling, three samples of mass spec grade pre-digested bovine serum albumin (BSA) were prepared at a concentration of 150pmol. These samples were included to account for differences in labelling efficiency between TMT batches. Three TMT batches were used to label the 36 experimental conditions, so three additional standards were prepared to be used for normalization. All samples, including the three BSA standards, were labelled using 3 sets of 13 tags from a 16plex TMT (Tandem Mass Tag) kit for quantification. Each BSA standard was labelled with a different tag. After combination and drying of all peptide samples, each combined TMT sample (including one BSA-labelled standard) 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 2.5. 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 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.