Fig. 4
From: Amyloid fibril proteomics of AD brains reveals modifiers of aggregation and toxicity
Multidimensional MS-based proteomic analyses identify amyloid fibril proteome. a Summary of MS analyses performed. b Experimental workflow using 15N-labeled brain tissue as a control to identify nonspecific co-purifying proteins. c Only a small panel of non-specific background proteins are identified in purified amyloid fibrils based on the identification of 14N and 15N labeled proteins; small inset shows identified 15N proteins. d The new purification strategy significantly reduces the number of proteins identified in P11 fractions compared to P6 fractions collected from App KI (AppNL/NL, AppNL−F/NL−F, and AppNL−G−F/NL−G−F) and transgenic 5xFAD brains. e Venn diagram depicting the number of proteins identified in purified fibril cores across the indicated mouse strains at a 1% protein FDR. f Venn diagram comparing proteins identified in fibrils isolated from control and AD human brains. g The GO-cellular components analysis with proteins identified in mouse (e) and human (f) amyloid fibrils. h Number of proteins identified in label-free MS analysis of amyloid fibril cores extracted from mouse brain tissues following digestion with multiple proteases. i Venn diagram comparing number of proteins identified across different mouse strains in multi-protease digestion (h) LC–MS/MS analysis. j Number of proteins identified in label-free MS analysis of fibril cores following multiprotease digestion of human brain-derived amyloid fibrils. k Venn diagram comparing number of proteins identified across human fibrils (j) digested with multiple proteases. l Scatter plots comparing average TMT intensities of AppNL−F/NL−F, and AppNL−G−F/NL−G−F with AppNL/NL fibril cores. Two biological replicates were pooled for each TMT channel. m Representative immunoblots confirming the presence of selected proteins identified in the proteomic analyses. n Representative dot blots for same proteins in AppKI amyloid fibrils. Data in c, d, h, and j represents mean ± SEM; *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001 analyzed with unpaired Student’s t-test or one-way ANOVA with post hoc Sidak test. P = pellet, and S = supernatant. NL = AppNL/NL, NL-F = AppNL−F/NL−F, NL-G-F = AppNL−G−F/NL−G−F. All mice were 6 months of age unless indicated. N = 4 mice (c), 4—8 mice (d and e), N = 15 control, 13 AD A2, 23 AD A3 humans (f), 7–8 mice (h, i), 10 humans (j, k)