Additional file 2 of Network analysis of the cerebrospinal fluid proteome reveals shared and unique differences between sporadic and familial forms of amyotrophic lateral sclerosis

Supplementary Material 2. Supplemental Fig. 1. a. Multidimensional scaling was used on raw data that had been log-transformed to visualize multidimensional proteomic distribution in two-dimensional space. b. Post-TAMPOR mode 3 distribution of TMT-MS data removes batch-level effect present in raw data. c. Post-regression TMT-MS data indicate that distribution is not affected by methodological artifacts. d. Raw DIA-MS data processed with log-transformation demonstrate the difference in unprocessed DIA and TMT-MS. e. Post-TAMPOR mode 4 distribution of individual points show a reduction in any clustering that may be due to DIA-MS. f. Post-regression distribution is further removed of methodical influence. Panels a-c demonstrate distribution of TMT-MS proteome and panels and d-f demonstrate distribution of DIA-MS. Each datapoint represents an individual tissue sample with colors indicating, in TMT-MS, shared batch and, in DIA-MS, shared center of origin. Supplemental Fig. 2. a. Shared and diverging differentially abundant proteins from single center TMT-MS quantification were compared between sporadic ALS and C9orf72 ALS. This scatterplot includes cases from the Emory single center dataset. Only proteins that were differentially abundant in both ALS subtypes are visualized. The number of proteins in each quadrant is denoted by “n”. b. Volcano plot showing differential abundance profiles comparing asymptomatic C9orf72 ALS (n = 10) and sporadic ALS (n = 35). Proteins that were significantly (p ≤ 0.05) down in disease (C9orf72 ALS, relative to control) are depicted in blue (n = 151), proteins that were significantly up are depicted in red (n = 139), and proteins that were neither significantly up nor down are grey. Supplemental Fig. 3. a. Volcano plot showing differential abundance profiles comparing asymptomatic C9orf72 carriers (n = 59) and controls (n = 72). Differentially abundant proteins were mapped by module. The height of the bars represents the fraction of module member proteins that were differentially abundant. The bars are color coded by heatmap for average log2 difference in abundance, where red represents an increase in abundance, and blue represents a decrease in abundance. b Asymptomatic SOD1 (n = 13) compared to controls. Differentially abundant proteins were also mapped by module. Log2 fold change (x-axis) and one-way ANOVA with Benjamini–Hochberg corrected by disease -log10 p-values (y-axis). Proteins significantly (p < 0.05) increased in abundance are depicted in red, significantly decreased in blue, and neither in grey. c. Differentially abundant proteins from asymptomatic C9orf72 HRE versus control were compared to asymptomatic SOD1 mutation carriers in a Venn diagram. Most robustly increased and decreased proteins are shown in red and blue boxes; respectively. Supplemental Fig. 4. a. Volcano plot representing differential protein abundance comparing SOD1 ALS (n = 22) versus control (n = 72). Log2 fold change (x-axis) and one-way ANOVA with Benjamini–Hochberg corrected by disease -log10 p-values (y-axis) are shown for each protein (n = 2,330). Proteins that were significantly (p ≤ 0.05) down in disease (SOD1 ALS, relative to control) are depicted in blue (n = 270), proteins that were significantly up are depicted in red (n = 186), and proteins that were neither significantly up nor down are grey. b. SOD1 protein abundance compared among all individuals without an SOD1 mutation, those with either SOD1A5 V or SOD1A5 T (including both symptomatic and asymptomatic carriers), and all other SOD1 mutation carriers including asymptomatic and symptomatic carriers. One-way ANOVA was used to determine if a difference was present between the SOD1-mutation groups. Note SOD1 protein is significantly reduced in those with a point mutation at position 5. c. SOD1 specific-peptide level quantification across controls and disease subgroups. Peptides were visualized for overlap of the canonical SOD1 protein sequence (P00441). Individual mutations are depicted with unique colors. Peptides in red come from DIA-MS, peptides in blue come from TMT-MS. d. Boxplots for abundance of each peptide identified were evaluated with one-way ANOVA. Each datapoint in the SOD1 ALS group is annotated with the mutation associated with each patient