Fig. 1. Free Nt-α-amino forms of Nt-acetylated proteins bind to IAPs in cells.

(A) Venn diagram summarizing numbers and percentages of all proteins with identified N termini from a large-scale MS experiment using HeLa cells (see experimental scheme in fig. S1). Total numbers and percentage of acetylated, dimethylated (reflecting unmodified, mono, and potentially also dimethylated N termini in cells), and proteins identified with both modifications are shown. All identified N termini are listed in detail in table S1. (B) Scheme of the performed peptide pull-down experiments. Nt-Ac or free Nt-α-amino (Nt-free) biotinylated peptides were bound to streptavidin beads. Beads were then incubated with HeLa lysates, washed, and directly digested on beads for MS analysis. Quantification was performed by using label-free intensities (label-free quantitation) or stable isotope labeling by amino acids in cell culture (SILAC). (C) Table of examples of N-terminal peptides from the MS dataset (A) used for pull-downs in 1(D to G) and fig. S2. Identified tryptic peptide, its N-terminal modification and posterior error probability (PEP) of the identification are specified. (D to I) Volcano plots from pull-down experiments comparing binders for free Nt-α-amino and Nt-Ac peptides from CDC20 (D), NIT1 (E), COMMD10 (F), and GOT1 (G) are shown. (H and I) The positive controls DIABLO and CASP9 are shown in (H) and (I), respectively. For all pull-downs the −log₁₀ of the t test values (y axis) are plotted against the log₂ of the fold difference of the t test (x axis) [t test, free Nt-α-amino (indicated as Nt-free) versus Nt-Ac; cutoff curve, false discovery rate (FDR) < 0.01, S0 > 2]. IAP family members are highlighted in green. LC-MS/MS, liquid chromatography–tandem MS.