Development of a label‒free nanolc/ms/ms assay for monitoring the changes in the proteomic landscape of thrombin‒activated human platelets

Abstract

One top research assay used to validate the efficacy of pharmacological agents used in antiplatelets aggregation therapy is mass spectrometry coupled with high throughput profiling of changes in the proteomic expression in the activated platelets exposed to inhibitors or agonists of platelets signaling. Herein we present the development of a new, bottom-up proteomics platform that enabled monitoring of changes in protein expression profiles of healthy human platelets, ex-vivo activated with thrombin, using as reference the proteome of resting platelets. The assay employed the platelets cryogenic lysis and protein solubilization under denaturant conditions, enabling the extraction of key membranebound proteins, in addition to the cytosolic and organelles-derived proteins secreted in microparticles, and found in the platelets releasates. Nano LC-ESI/MS/MS sequencing of tryptic/Glu-C/Lys-C peptides from the "in solution" one step digestion of the whole platelets proteomes (2x10 8 platelets/patient sample) was performed on a Q-Exactive quadrupole orbitrap mass spectrometer coupled with label free quantification (LFQ) analysis. The changes in proteomic landscapes were qualitatively and quantitatively analyzed using a combination of systems biology and bioinformatics approaches empowered by the ingenuity pathway analysis (IPA), and the screening of identified proteins and genes entities against curated databases containing platelets proteomes, including the "Adhesome", "Exocarta", "Reactome" and "Platelet Web". The label-free global proteomics analysis retrieved a total of 924 proteins (FDR <1.3% for proteins and <0.8% for peptides) out of which 330 were shared between resting and thrombin-activated platelets. About 50% of the total proteome of thrombin-activated platelets was represented by up-regulated and previously validated protein biomarkers, involved in the pathways mediating the protease and thrombin activated receptor (PARs), integrin signaling, the actin and rhoA linked to cytoskeleton signaling, and activated kinases pathways regulating the cellular motility, aggregation, procoagulation and degranulation. In conclusion, the systems biology approaches validated our newly developed LFQ proteomic assay as a reliable tool for monitoring the changes in protein expression profiles in thrombin-activated platelets, and further advocate for employment of these approaches in assessing the efficacy of antiplatelets drugs (inhibitors of platelets aggregation) during the prevention and treatment of cardiovascular and cerebrovascular diseases. Citation: Gonzalez J, Babinska A, Ewul ELV, et al. Development of a label-free nanolc/ms/ms assay for monitoring the changes in the proteomic landscape of thrombin-activated human platelets. MOJ Proteomics Bioinform. 2018;7(4):232-255. Platelets purification and ex-vivo activation with thrombin for proteomic analysisPRP were subjected to the whole platelets purification as described previously by other research groups. [30][31][32][33][34][35] Then, platelets were pelleted at 750xg ...