Characterization of N-terminal modifications in platelets. (A) Distribution of platelet N termini as natural (as synthesized or after maturation) and internal (proteolysis-derived neo-N termini) termini among the 2960 high-confidence positionally annotated peptides identified in total by TAILS and pre-TAILS analyses. Fractions of identified Nα-acetylated (Ac) vs free (which become iTRAQ labeled in TAILS protocol) N termini within each category are indicated. Frequency distribution of N-terminal processing after removal of the initiator methionine, propeptides, and signal peptides is shown. (B) Distribution of natural and internal N termini among 2290 free and 670 acetylated peptides among 2960 high-confidence positionally annotated peptides. (C) Amino acid frequency distribution in P2′ position following intact initiator methionine [ie, (Ac)M-X)] (acetylated or not) for natural N termini (open bars) vs internal N termini (black bars). (D) Amino acid frequency distribution in P1′ position (acetylated or not) following initiator methionine excision [ie, M.(Ac)X] in natural N termini (open bars) vs internal N-termini (black bars). (E) Amino acid distribution in P2′ position following intact initiator methionine in acetylated (ie, AcM-X) natural N-termini (open bars) vs internal N termini (black bars). (F) Amino acid frequency distribution in P1′ position following initiator methionine excision for acetylated (ie, M.AcX) natural N termini (open bars) vs internal N termini (black bars). Gray bars show amino acid frequency distribution in P1′ position for acetylated internal N termini lacking putative initiator methionine in P1 position (ie, Z.AcX, where Z is any amino acid other than methionine). Number of peptides in each category is indicated in brackets. *Indicates N termini that are potential products of translation at alternative start sites predicted based on their N-terminal amino acid distribution analysis.