Extensive proteolytic processing in human platelets during storage. Platelets were stored under blood-banking conditions for 9 days with or without EDTA-free cOmplete protease inhibitor cocktail, and the platelet proteome from different days was characterized by 4-plex iTRAQ TAILS (day 2, day 4, day 4 + inhibitors [Inh], day 9). Of 1878 high-confidence peptides identified by TAILS, 1102 were quantified and had positional annotation in UniProt/Swiss-Prot. (A) Distribution of log2 iTRAQ (day 4/day 4+Inh) ratios for 414 natural N termini identified in 4-plex TAILS analysis. Two times the standard deviation corresponding to the ratios of 2 and 0.5 were chosen as statistical significance ratio cutoffs and are indicated by a dashed line. Low iTRAQ (D4/D4+Inhibitors) ratios (≤0.5) of natural N termini indicate decrease in their abundance due to proteolysis in the absence of inhibitors. (B) Distribution of log2 iTRAQ (D4/D2) ratios for internal N termini identified in 4-plex TAILS analysis. The statistical significance cutoffs of 2 and 0.5 are indicated by dashed lines. High iTRAQ (D4/D2) ratios (≥2) indicate increased production of internal neo-N-terminal peptides due to proteolytic processing in the absence of inhibitors. N termini found with intermediate iTRAQ ratios (0.5 ≥ ratios ≤ 2) represent products of proteolysis that are not changing in time. (C) Quantitative overview of the 481 N-terminal peptides that were significantly up- (ratios ≥2; right side of the graph) or downregulated (ratios ≤0.5; left side of the graph) in at least 1 comparison (Day 4/Day 2, Day 9/Day 2, or Day 4/Day 4+Inh). Percentile fractions of internal N termini (black) and natural N termini (white) among downregulated or upregulated N termini are shown. Total number of peptides in each category is indicated. (D) Fold enrichment of internal (black bars) vs natural (white bars) N termini for significantly up- (ratios ≥2; right side) or downregulated (ratios ≤0.5; left side) N termini as summarized in Figure 3C. For each category, percentile distribution of internal and natural N termini was normalized for the total levels of natural and internal N termini (ie, 38% and 62%, respectively) observed in the subset of 1102 quantifiable peptides and expressed as log2 ratio.