Effect of exogenous broad-spectrum metalloproteinase inhibitor on platelet N-terminome during storage. Platelets were stored under blood-banking conditions for 7 days in the absence or presence of 10 μM marimastat. Two technical replicates were performed. Platelet proteomes from different time points and conditions were analyzed by 8-plex iTRAQ-TAILS analyses. Out of 1999 high-confidence peptides identified by TAILS in 2 technical replicates, 1111 could be quantified and had positional annotation in the UniProt/Swiss-Prot database. (A) Quantitative overview of the 178 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 response to marimastat on at least 1 sampling day (ie, as indicated by iTRAQ_{DMSO/Marimastat} on days 3, 5, and 7). Percentile fraction of internal or neo-N termini (black bars) and natural N termini (white bars) is shown. Number of peptides in each category is shown. (B) Fold enrichment of neo- (black bars) vs natural (white bars) N termini for significantly up- (ratios ≥2; right side of the graph) or downregulated (ratios ≤0.5; left side of the graph) N termini are shown. For each category, percentile distribution of internal and natural N termini was normalized for the total levels of these N termini observed among a total of 1111 quantifiable and positionally annotated peptides (ie, 31% and 69% of natural and internal N termini, respectively) and expressed as a log2 ratio. (C) Western blot validation of metalloproteinase substrates identified by TAILS: tubulin, ρ-GDI, gelsolin, talin. Protein expression was evaluated in 2 individual platelet units in at least 2 technical replicates, and representative blots are shown. Arrows and arrowheads indicate position of the full-length protein and cleavage products, respectively.