Figure 3.

Global protocol of combined proteome analysis of (un)stimulated platelets. (A), Human platelet isolation from freshly drawn blood. Platelet-rich plasma (PRP) is collected by centrifugation and then re-centrifuged twice to obtain double-washed platelets. Removal of remaining leukocytes’ antibody-coated beads is advised. Platelet purity is determined by flow cytometry or microscopy. (B), Isolated platelets are stimulated with agonists as required. After lysis, proteins are fragmented by trypsin under well-controlled conditions, in this case in the presence of unique stable isotope labels. Pooled, labelled samples are fractionated, and peptides are resolved by LC-MS/MS analysis. For quantitative proteome information, reference peptides can be added. In case of phosphoproteome analysis or other post-translational modifications, an enrichment step is included for improved detection. An example protocol is provided in Box 1. Created with BioRender.com.