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.
Sample Processing Protocol
Enriched granule fractions were suspended in a surfactant containing ammonium bicarbonate buffer (Rapigest, Waters, Milford, USA) (pH 8.2) and homogenized according to the manufacturer's instructions. Protein concentration was measured by Bradford quantification (BioRad, Hercules, USA). Fifteen micrograms of protein from each sample was then reduced using dichlorodiphenyltrichloroethane (5 mM) and alkylated by adding iodoacetamide (15 mM). Trypsin (1:20 weight/weight) was added and incubated overnight. Rapigest was then removed according to the manufacturer's protocol. Finally, samples were desalted using a C18 microspin column (Harvard, Holliston, USA) according to manufacturer's instructions.
Data Processing Protocol
Protein identification was performed using the EasyProt resource. Briefly, the EasyProt 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. Homo sapiens taxonomy was specified for database searching. The parent ion tolerance was set to 10 ppm. Variable amino acid modifications were oxidized methionine. Trypsin was selected as the enzyme, with one potential missed cleavage, and the normal cleavage mode was used. Only one search round was used with selection of “turbo” scoring. The peptide p value was 1 E-2 for LTQ-OT data. False-positive ratios (FDR) were estimated using a reverse decoy database. All datasets were searched once in the forward and once in the reverse database. Separate searches were used to keep the database size constant. Protein and peptide scores were then set to maintain the FDR below 1%. This resulted in an only slight overestimation of the false-positive ratio. For all analyses, only proteins matching two different peptide sequences were kept.
Zufferey A, Schvartz D, Nolli S, Reny JL, Sanchez JC, Fontana P. Characterization of the platelet granule proteome: Evidence of the presence of MHC1 in alpha-granules. J Proteomics. 2014 Feb 16. pii: S1874-3919(14)00057-8 PubMed: 24549006