Deciphering changes to human platelet proteome and degradome during storage before transfusion

Proteases, and specifically metalloproteinases, have been linked to the loss of platelet function during storage before transfusion, albeit the mechanism remains unknown. We used a dedicated N-terminomics technique, multiplex iTRAQ-TAILS (Terminal Amine Isotope Labeling of Substrates), to characterize the human platelet proteome, N-terminome, and their post-translational modifications throughout platelet storage under blood banking conditions. From the identified 2,938 proteins and 7,503unique peptides we characterized N-terminal methionine excision, co- and post-translational N-acetylation, maturation and proteolytic processing of proteins in human platelets. We also identified for the first time in platelets 10 proteins previously classified as “missing” in the human proteome. Most of identified N-termini (77%) were internal, of which 105 were novel potential alternative translation start sites, with 2,180 representing stable proteolytic products, thus highlighting a prominent previously uncharacterized role of proteolytic processing during platelet storage. Protease inhibitor studies revealed metalloproteinases as being primarily responsible for proteolytic processing (as opposed to degradation) during storage. System-wide identification of metallo- and other proteinase substrates and their respective cleavage sites offers novel potential mechanisms of their effect on protein activity and platelet function during storage.