Fig. 1: nanoLC–MS/MS reveals increased protein ubiquitination in the ipsilateral cortex after cerebral ischemia.

a Schematic of the nanoLC–MS/MS experimental workflow. Mice underwent either sham or MCAO/1 h reperfusion surgeries, and sham, ipsi- and contralateral cortices were extracted (a). Triton-insoluble proteins were isolated (b) and digested with trypsin (c). Peptides with- and without K-ε-GG peptide enrichment (d) were subjected to nanoLC–MS/MS and downstream data analysis (e). n = 3 MS runs for sham and ipsilateral, n = 2 MS runs for contralateral (including each 20 mice/group). b Confirmation of increased protein ubiquitination after ischemia in detergent-insoluble fractions throughout the entire cerebral cortex. Ubiquitination was assessed by Western Blotting with anti-ubiquitin antibody in sham and ipsilateral cortical areas used for proteomic analyses. Antibody against β-tubulin served as loading control. n = 1–2 mice/group. c Graphs showing the count and abundance of global and ubiquitinated peptides in sham (s1–3), ipsi- (i1–3), and contralateral (c1–2) Triton-resistant samples across MS experiments. Gray boxes indicate the percentage of peptide hits with counts greater than 250 per abundance bin higher than log₁₀ = 5 (for global peptides) or greater than 50 per abundance bin higher than log₁₀ = 6 (for Ub peptides). d Heatmap of hierarchical clustering of ubiquitinated peptide abundances across experimental groups and MS runs. e Heatmap of hierarchical clustering of ubiquitinated protein abundances across experimental groups and MS runs. c contralateral, ctx cortex, hr hour, i ipsilateral, K-ε-GG diglycyl-lysine, MCAO middle cerebral artery occlusion, n.t. not tested, rep reperfusion, s sham, Tx Triton X100, Ub ubiquitin.