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Simplified platelet sample preparation for SDS ‐ PAGE ‐based proteomic studies
Author(s) -
Reicheltová Zuzana,
Májek Pavel,
Riedel Tomáš,
Suttnar Jiří,
Dyr Jan E.
Publication year - 2012
Publication title -
proteomics – clinical applications
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.948
H-Index - 54
eISSN - 1862-8354
pISSN - 1862-8346
DOI - 10.1002/prca.201100101
Subject(s) - coomassie brilliant blue , centrifugation , chromatography , chemistry , platelet , staining , proteome , stain , spots , trypsin , platelet rich plasma , microbiology and biotechnology , andrology , biochemistry , biology , medicine , pathology , immunology , enzyme
Purpose The goal of this study was to design an easy and simple protocol for platelet isolation and sample preparation for proteomic studies based on 2 DE ( IEF – SDS ‐ PAGE ) followed by Coomassie blue staining. Experimental design Blood was collected by venipuncture into tubes coated with EDTA and platelet‐rich plasma ( PRP ) was immediately obtained by centrifugation. PRP was stored refrigerated in closed Falcon tubes for 0, 1, 2, 3, 5, and 7 days and platelets were isolated by centrifugation. 2 DE gels were stained with colloidal Coomassie blue stain and evaluated using the Progenesis SameSpots software. Spots that differed significantly in the gels of fresh and stored platelet samples were excised, digested with trypsin, and further analyzed using nano LC ‐ MS / MS . Results During the 7‐day follow‐up period, we found 20 spots that differed significantly ( ANOVA p <0.05). During the first 2 days of PRP storage in test tubes, however, only nine spots significantly differed in all donors. In these spots, we identified 14 different proteins. Conclusions and clinical relevance In conclusion, for proteome investigations, whenever it is not feasible to prepare washed platelets immediately after blood collection, the EDTA ‐anticoagulated PRP can be stored in test tubes at 4°C for up to 2 days for the platelet proteome investigation.