400mg/L) followed by dissection. The tissue was collected from the optic nerve head to the optic chiasm. The tissues were immediately frozen on dry ice. Optic nerve samples were pooled for each category (female crush, female control, male crush, male control) and pooled at n = 31 to obtain sufficient protein concentrations for analysis (pooled optic nerves served as one sample). Retina and tectum samples were pooled using the same categories (female crush, female control, male crush, male control) at n = 10-12 (pooled tissue served as one sample). Protein extraction was carried out by homogenizing the optic nerve tissue in TEAB, NaCl and SDS. Three synthetic 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 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 2 sets of 14 tags from a 18plex TMT (Tandem Mass Tag) kit (A52045: Thermo Fisher Scientific, Waltham, MA) for quantification. After combination and drying of all peptide samples, the samples were fractionated into 9 fractions using Pierce High pH Reversed-Phase Peptide Fractionation Kit (84868: Thermo Fisher Scientific, Waltham, MA). After fractionation and drying of all peptide samples, each fractionated TMT sample was spiked with two additional peptide standards (Regen II) containing isobaric labels. The final concentration of the post extraction spiked peptides was 54uM per plex. These standards (Regen II) were spiked 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. Untargeted liquid chromatography-mass spectrometry was performed on an Easy-nLC 1000 liquid chromatograph coupled to a QExactive mass spectrometer (LC-MS/MS). Raw mass spectrometry data files were analyzed using Proteome Discoverer 3.0. The Danio rerio 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. Two additional local databases were created for the pre- and post- extraction peptides and ran separately from experimental protein identification.]]>