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TAILS N-terminomics of human platelets reveals pervasive metalloproteinase dependent proteolytic processing in storage

Anna Prudova, Katherine Serrano, Ulrich Eckhard, Nikolaus Fortelny, Dana V. Devine and Christopher M. Overall

Key points

  • TAILS proteomics identified 2,938 human platelet proteins, pervasive proteolytic processing and precise proteolytic cleavage sites in stored platelets.

  • Metalloproteinase activity strongly generated protein cleavage products in the platelet storage lesion that was reduced with a MMP inhibitor.

Abstract

Proteases, and specifically metalloproteinases, have been linked to the loss of platelet function during storage before transfusion, however, the underlying mechanisms remain unknown. We used a dedicated N-terminomics technique, iTRAQ-TAILS (Terminal Amine Isotopic Labeling of Substrates), to characterize the human platelet proteome, N-terminome, and post-translational modifications throughout platelet storage over 9 days under blood banking conditions. From the identified 2,938 proteins and 7,503 unique peptides we characterized N-terminal methionine excision, co- and post-translational Nα-acetylation, protein maturation and proteolytic processing of proteins in human platelets. We also identified for the first time in platelets 10 proteins previously classified by HUPO as "missing" in the human proteome. Most of N-termini (77%) were internal neo-N-termini: 105 were novel potential alternative translation start sites; and 2,180 represented stable proteolytic products, thus highlighting a prominent yet 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 suggests novel mechanisms of the effect of proteases on protein activity and platelet function during storage. All datasets and metadata are available through ProteomeXchange with the dataset identifier PXD000906.

  • Submitted April 11, 2014.
  • Accepted October 1, 2014.