Institute of Hematology and Blood Transfusion, Prague, Czech Republic.
Proteomics Clin Appl. 2012 Aug;6(7-8):374-81. doi: 10.1002/prca.201100101.
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.
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.
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.
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.
本研究旨在设计一种简单易行的方案，用于基于 2-DE（IEF-SDS-PAGE）后考马斯亮蓝染色的蛋白质组学研究中血小板的分离和样品制备。
通过静脉穿刺将血液采集到涂有 EDTA 的管中，并立即通过离心获得富含血小板的血浆（PRP）。将 PRP 在封闭的 Falcon 管中冷藏保存 0、1、2、3、5 和 7 天，并通过离心分离血小板。使用胶体考马斯亮蓝染色对 2-DE 凝胶进行染色，并使用 Progenesis SameSpots 软件进行评估。从新鲜和储存的血小板样品凝胶中差异显著的斑点被切除、用胰蛋白酶消化，并进一步使用纳升液相色谱-串联质谱（nanoLC-MS/MS）进行分析。
在 7 天的随访期间，我们发现有 20 个斑点差异显著（ANOVA p <0.05）。然而，在管中储存 PRP 的头 2 天，所有供体中只有 9 个斑点差异显著。在这些斑点中，我们鉴定出 14 种不同的蛋白质。
总之，对于蛋白质组学研究，只要在采集血液后不能立即制备洗涤血小板，EDTA 抗凝的 PRP 可以在 4°C 的试管中储存长达 2 天，用于血小板蛋白质组学研究。
