Characterization of the proteome of human blood platelet sercretory granules using gas-phase fractionation (GPF)

We performed an extensive qualitative characterization of the platelet granule proteome using subcellular fractionation followed by mass spectrometry analysis and functional annotation. Platelets were isolated from whole blood by centrifugation and their level of purity was tested by microscopic inspection, western blot and Leucocount kit. Subcellular fractionation based on sucrose gradient was used to enrich sample in secretory granules. Then sample quality was assessed by western blot and electronic microscopy, prior proteomic study. Samples were trypsin digested and analyzed using an Orbitrap Velos in a gas-phase fractionation mode. The m/z ranges for the selection of precursor ions were 400-521, 516-690, 685-986 and 963-2000 Thomsons. The 2 technical replicates were analyzed by a GPF series of injections. Protein identification was performed using the EasyProt resource. The module EasyProtConv was used to generate the MGF file from the raw data. The peaklist files were searched against the UniProt database (2011_02 of 08-Feb-2011) using EasyProt. Eight-hundred-and-twenty-seven proteins were identified, most of them being associated to granules and to the granule’s secretory machinery.