Protein biomarkers of external root resorption: A new protein extraction protocol. Are we going in the right direction?

OBJECTIVE: The aim of this study is to determine a protocol of gingival crevicular fluid protein extraction used for the first dimension of 2-DE gels. It also aims at conducting a review on the current candidates for protein markers of this pathology, all of which may be used to prevent the disease.METHODS: Gingival crevicular fluid was collected from two groups of 60 patients each, with and without external root resorption. Samples were extracted by means of various methods of protein extraction. SDS-PAGE gels were used to assess the quality of the method which was subsequently tested during isoelectric focusing of 2-DE gels taken from samples of patients with and without the disease.RESULTS: Milli-Q ultrapure ice cold water, without precipitation for gingival crevicular fluid protein extraction, proved the method with greatest sharpness to detect protein bands. Additionally, it allowed two-dimensional electrophoresis to be performed.CONCLUSION: The new protein extraction protocol does not interfere in isoeletric focusing of 2-DE gels. Furthermore, it provides the greatest sharpness in detecting protein bands of SDS-PAGE gels. This will allow mapping and searching of new external root resorption markers, particularly due to the difficulty in carrying out molecular tests with the current candidates for protein markers.

研究目的：本研究旨在建立适用于双向凝胶电泳（two-dimensional electrophoresis, 2-DE）第一向电泳的牙龈沟液（gingival crevicular fluid, GCF）蛋白质提取方案，并对当前该疾病的蛋白质标志物候选物进行综述，所有候选物均可用于该疾病的预防。
研究方法：本研究从各60例的两组患者（分别伴或不伴外源性牙根吸收）中采集牙龈沟液样本，采用多种蛋白质提取方法对样本进行处理；通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE）评估各提取方法的质量，随后将优化后的方法应用于伴/不伴该疾病的患者样本的2-DE凝胶等电聚焦实验中进行验证。
研究结果：使用Milli-Q超纯冰冷水、无需沉淀步骤的牙龈沟液蛋白质提取方法，在检测蛋白质条带时表现出最高的清晰度，且可顺利开展双向凝胶电泳实验。
研究结论：本研究建立的新型蛋白质提取方案不会干扰2-DE凝胶的等电聚焦过程，同时可使SDS-PAGE凝胶的蛋白质条带检测清晰度达到最优。鉴于当前已报道的蛋白质标志物候选物在分子检测中存在较大难度，本方案将为外源性牙根吸收相关新型标志物的图谱绘制与筛选工作提供有力支撑。