Fig. 1: Quantitative proteomic profiling for NEDD4 regulated proteins and molecular pathways.
a Workflow for analysis of ubiquitin remnant enriched and pre-enrichment peptides. SILAC labelled NCU10K cells treated with low GC (control, light - red) or Nedd4 (heavy, green) siRNA were lysed in 8 M urea, equal amounts (5 mg each) were combined and digested with trypsin. The resulting peptides were fractionated by offline basic reversed-phase HPLC into 80 fractions that were then pooled in a discontinuous pooling strategy into 8 fractions. The bulk of each fraction was then subjected to ubiquitin remnant (K-ε-GG remnant) enrichment and enriched peptides identified and quantified by LC-MS/MS. A small amount of each fraction was also analysed prior to enrichment for total protein identification and quantification for use in subsequent normalisation and data analysis. b The combined number of quantified protein groups (total number observed in brackets) and Venn diagram showing overlap of protein group IDs from the total protein (unenriched) fractions across 2 biological replicates (rep). c Correlation of log2 H/L ratios for each protein group common to both replicates (R2 = 0.7156). d Frequency distribution of log2 H/L ratios for protein groups with a mean of −0.07 and standard deviation of 0.596. e Correlation of total protein abundance (Y-axis) to gene expression data (X-axis) (R2 = 0.21) for proteins with altered abundance. Proteins indicated in green had a statistically significant reduction in gene expression (step-up p < 0.05). Proteins indicated in magenta had a statistically significant increase in gene expression (step-up p < 0.05) as determined by two-sided ANOVA with adjustments made using Benjamini-Hochberg Step-Up controlling procedure. f Top 10 annotations from process network analysis of differentially expressed genes at the mRNA level using DAVID. Fold enrichment and p-values derived using Fisher’s exact test with adjustments using Bonferroni, Benjamini and FDR methods are shown.
