Improving the Proteomic Analysis of Archival Tissue by Using Pressure-Assisted Protein Extraction: A Mechanistic Approach.
Improving the Proteomic Analysis of Archival Tissue by Using Pressure-Assisted Protein Extraction: A Mechanistic Approach.
复制标题
通过使用压力辅助蛋白质提取改进档案组织的蛋白质组分析：一种机械方法。
DOI：
10.4172/jpb.1000315
复制
发表时间：
2014-06-24
期刊：
影响因子：
--
通讯作者：
Mason JT
中科院分区：
文献类型：
--
作者：
Fowler CB;O'Leary TJ;Mason JT
甲醛固定，石蜡包埋（FFPE）组织库代表了疾病进展和治疗反应的回顾性研究的宝贵资源。然而，FFPE组织的蛋白质组学分析受到甲醛诱导的蛋白质修饰的阻碍，这降低了蛋白质提取效率并可能导致蛋白质错误鉴定。在这里，我们证明了使用高静水压力（40，000 psi）作为一种新的方法，从FFPE组织的完整的蛋白质的恢复增加的热量。我们的实验室采取了一种机械方法来开发改进的蛋白质提取方案，首先研究甲醛与蛋白质的反应以及逆转这些反应的方法，然后将这种方法应用于称为“组织替代物”的模型系统，该模型系统是通过用甲醛处理高浓度的细胞质蛋白质形成的凝胶，最后是FFPE小鼠肝脏组织。我们的研究表明，升高的压力通过水合和促进高度聚集的蛋白质的溶解，从而允许随后逆转（通过水解）甲醛诱导的蛋白质加合物和交联，从而提高了FFPE组织替代物中蛋白质的回收率。当使用加热和高压提取FFPE小鼠肝脏时，与仅用加热提取的匹配组织相比，蛋白质提取效率增加了4倍，通过质谱鉴定的非冗余蛋白质数量增加了30倍。更重要的是，在FFPE组织中鉴定的非冗余蛋白质的数量与相应的冷冻组织几乎相同。
Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE tissue. Our laboratory has taken a mechanistic approach to developing improved protein extraction protocols, by first studying the reactions of formaldehyde with proteins and ways to reverse these reactions, then applying this approach to a model system called a “tissue surrogate”, which is a gel formed by treating high concentrations of cytoplasmic proteins with formaldehyde, and finally FFPE mouse liver tissue. Our studies indicate that elevated pressure improves the recovery of proteins from FFPE tissue surrogates by hydrating and promoting solubilization of highly aggregated proteins allowing for the subsequent reversal (by hydrolysis) of formaldehyde-induced protein adducts and cross-links. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency and up to a 30-fold increase in the number of non-redundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of non-redundant proteins identified in the FFPE tissue was nearly identical to that of the corresponding frozen tissue.