UBC Theses and Dissertations
Application of proteomics to the study of protein translation in stored platelet units Thon, Jonathan Noah
Platelet products have a short shelf life (5 to 7 days) owing in part to the deterioration of the quality of platelets stored at 22°C. This creates significant inventory challenges, and blood banks may suffer shortages and high wastage as a result. Proteomics offers a global quantitative approach to investigate changes occurring in stored blood products. These data sets can identify processes leading to storage-associated losses of blood component quality such as the platelet storage lesion (PSL). Changes to the platelet proteome between days 1 and 7 of storage were analysed with 3 complementary proteomic approaches with final mass spectrometric (MS) analysis: 2-dimensional (2D) gel electrophoresis/differential gel electrophoresis (DIGE), isobaric tagging for relative and absolute quantification (iTRAQ), and isotope-coded affinity tagging (ICAT). Although proteomics analyses identified many storage-associated protein changes, these varied significantly by method suggesting that a combination of protein-centric (2D gel or DIGE) and peptide-centric (iTRAQ or ICAT) approaches is necessary to acquire the most informative data. Validation of the proteomics results by western blotting, flow cytometry, quantitative real-time polymerase chain reaction (qRT PCR) and ³ٰ⁵S-methionine incorporation confirmed that platelets are capable of synthesising biologically relevant proteins ex vivo throughout a 10-day storage period with particularly long-lived mRNA (half-life of approximately 2.4 days), and has provided the first evidence for one of the mechanisms of the PSL. The development of an ³ٰ⁵Smethionine assay has since shown that stored human blood platelets incorporate ³ٰ⁵S-methionine at a rate that is proportional to time and substrate concentration, and is slower for freshly drawn platelets than those stored in pooled buffy coat derived units for 10 days. More interesting still are the observations that the overall ³ٰ⁵S-methionine incorporation rate was higher in pooled buffy coat platelet units versus freshly drawn platelets, that this rate increased upon agonist exposure in both, and that day 8 platelets showed significantly greater total protein translation than on days 2,3,7 and 10 of storage. This may be indicative of translational regulation of the platelet proteome during storage and upon activation. Translational control is a consequence of remarkable cellular specialisation and precise biochemical pathways which, in the case of platelets, may lead to storage-associated losses of blood component quality and must be understood if platelet storage times are to be extended.
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