Differential quantitation of 2560 proteins in the yeast WT/ nat3Δ proteome enables identification of NatB substrates and reveals overall increased phosphorylation levels. Panel (A), (C) and (D) display peptide and protein 15N/14N ratios (2log transformed) determined in both biological replicates. Data of the two biological replicates are plotted versus each other. In experiment 1 the ΔNat3 strain was labeled with 14N while WT incorporated the heavy 15N label. In experiment 2 the isotope labels were reversed. The dashed lines represent a 95% confidence interval indicating high reproducibility of ratio data between biological replicates . The circles indicate the chosen arbitrary thresholds for diminished or elevated protein levels, which were set at a three-fold change. Panel (A) displays 15N/14N ratio data of N-acetylated peptides, red colored spots mark N-acetylated peptides displaying the NatB target sequence while the lighter red indicates peptides located outside the 95% confidence. Panel (B) displays 15N/14N ratio histograms. The upper histogram shows ratios for all detected N-acetylated peptides not containing the expected NatB substrate sequence. The lower plot illustrates the ratio distribution of N-acetylated peptides containing the expected NatB substrate sequence, namely a methionine at the ultimate and an aspartic acid, glutamic acid or an asparagine in the penultimate position. Individual ratios from the biological replicates were averaged. The insets show frequency plots of the amino acids in the first 5 positions of the N-terminus generated by Weblogo. Panel (C) displays protein ratios as determined from unmodified peptides, with in red again the observed NatB substrate proteins. (D) displays phosphopeptide ratios, irrespective of being NatB substrate or not.