Simplified platelet sample preparation for SDS-PAGE-based proteomic studies.
OBJECTIVE
The goal of this study was to design an easy and simple protocol for platelet isolation and sample preparation for proteomic studies based on 2DE (IEF-SDS-PAGE) followed by Coomassie blue staining.


METHODS
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. 2DE 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 nanoLC-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
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.
Institute of Hematology and Blood Transfusion, Prague, Czech Republic. zuzana.reicheltova@uhkt.cz
Zuzana
Z
Reicheltová
Pavel
P
Májek
Tomáš
T
Riedel
Jiří
J
Suttnar
Jan E
JE
Dyr
D053000:Analytic Sample Preparation Methods; D016718:Arachidonic Acid / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D010974:Platelet Aggregation / Q000187:drug effects; D053657:Platelet-Rich Plasma / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D018179:Receptors, Thrombin / Q000378:metabolism
D018179:Receptors, Thrombin; D016718:Arachidonic Acid