Quantitative analysis of ADP receptor activation of human platelets

Human platelets play a key role in mediating homeostasis but also thrombosis. During activation, human platelets undergo dramatic shape change which is controlled by various phosphorylation-dependant pathways. In this project, we investigate the impact of ADP and prostaglandin-mediated stimulation on the phosphorylation patterns in human platelets at different time points of stimulation. In order to gain a global overview of phosphorylation changes we used iTRAQ quantitation with a subsequent optimized TiO2 enrichment after sample preparation. Since phosphorylation analysis is trace analysis, each step of the protocol has been checked via a robust quality control (QC) of the lysis, proteolytic digest and LC-MS system. Consequently, the TiO2 workflow was optimized and tested for reproducibility with errors of less than 15 % according to signal intensity of spiked-in standard peptides.

Currently, we quantified about 650 different phosphorylated peptides from human platelets. Of these, about 15 % show a significant (x2) up/down regulation compared to the control after ADP or Iloprost stimulation. Interesting peptide candidates (~300) have been chosen for peptide synthesis to set up a targeted mass spectrometry method based on Selected Reaction Monitoring (SRM). Thus, phosphorylated peptides and their non-phosphorylated counterparts can be directly quantified from cell lysates. Thus, accurate quantitation of a large set of biological replicates enables sound systems biology modelling.

In the long term, this study will improve our understanding of platelet activation/inhibition and thus may lead to new diagnostic tools for bleeding disorders.