Characterization of platelet proteome and degradome during storage. (A) Experimental setup. Blood was collected from healthy volunteers on day 0. Platelet units were prepared using buffy coat method and according to Canadian Blood Services blood-banking protocols on day 1. After resting, platelet units were subdivided into smaller units and vehicle or protease inhibitors were added. The resulting platelet units were stored for 9 days under the blood-banking conditions at 22°C with agitation. Platelet aliquots were sampled throughout storage. Platelets were washed, lysed, and subjected to proteomic analyses using TAILS and pre-TAILS approaches (similar to shotgun analysis) to yield the platelet N-terminome and proteome, respectively. In total, 4 TAILS analyses and 1 pre-TAILS analysis were performed. (B) Overlap in the number of total peptides (left) and proteins (right) identified by pre-TAILS and TAILS analyses. (C) Distribution of internal and N-terminal peptides among 5648 and 1878 peptides identified before (left) and after (right) N-terminal enrichment in 4-plex pre-TAILS and TAILS analyses of the same sample, respectively. N-terminal peptides originally present in the sample include naturally blocked (by Nα-acetylation) and free N termini. Free N termini become iTRAQ-labeled in TAILS protocol unless cyclization renders these unreactive (cyclization occurs with peptides with N-terminal glutamine, asparagine, and carbamidomethylated cysteine residues). The unlabeled peptides represent internal peptides released by trypsin during the proteome digest prior MS analysis. (D) Western blot validation of 2 “missing” proteins in the human proteome identified proteomically: nuclear pore membrane glycoprotein 210-like (Nup210L; left panel) and aquaporin-6 (right panel). Protein expression in 2 individual platelet units is shown. Arrows indicate different positions of the glycosylated and oligomerization forms of aquaporin-6 as previously assigned.³¹