Application of 2-D DIGE to formalin-fixed, paraffin-embedded tissues
Application of 2-D DIGE to formalin-fixed, paraffin-embedded tissues
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DOI：
10.1002/pmic.201000353
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发表时间：
2011-03-01
期刊：
影响因子：
3.4
通讯作者：
Addis, Maria Filippa
中科院分区：
文献类型：
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作者：
Tanca, Alessandro;Pagnozzi, Daniela;Addis, Maria Filippa
研究福尔马林固定、石蜡包埋（FFPE）组织的蛋白质组的能力可以被认为是临床蛋白质组学领域的一项重大成就。然而，基于凝胶的FFPE组织蛋白质组的研究方法已经落后，主要是因为全长蛋白质提取物的质量不足。在这里，2-D DIGE技术的适用性FFPE蛋白，重复FFPE提取物之间的内部再现性，以及FFPE和新鲜冷冻组织之间的可比性进行了研究。在重复FFPE组织提取物的标记和电泳后获得的2-D DIGE图案具有高度可重复性，具有令人满意的分辨率和复杂性。此外，DIGE的实施能够突出和表征FFPE谱与新鲜冷冻谱相比的一致差异，其由与蛋白质pI值直接相关的酸性位移和与赖氨酸残基的分子量和百分比直接相关的斑点信号强度的降低表示。在所有FFPE组织提取物重复中以相似的程度持续且可重复地存在，这些修饰似乎并不妨碍通过2-D DIGE对FFPE组织提取物进行比较分析，从而为其应用于档案组织库的差异蛋白质组学研究开辟了道路。
The ability to investigate the proteome of formalin-fixed, paraffin-embedded (FFPE) tissues can be considered a major recent achievement in the field of clinical proteomics. However, gel-based approaches to the investigation of FFPE tissue proteomes have lagged behind, mainly because of insufficient quality of full-length protein extracts. Here, the 2-D DIGE technology was investigated for applicability to FFPE proteins, for internal reproducibility among replicate FFPE extracts, and for comparability between FFPE and fresh-frozen tissue profiles. The 2-D DIGE patterns obtained upon labeling and electrophoresis of replicate FFPE tissue extracts were highly reproducible, with satisfactory resolution and complexity. Moreover, the implementation of DIGE enabled to highlight and characterize the consistent differences found in the FFPE profiles compared with fresh-frozen profiles, represented by an acidic shift, directly correlated to the protein pI value, and by a reduction in spot signal intensity, directly correlated to molecular weight and percentage of lysine residues. Being constantly and reproducibly present in all FFPE tissue extract replicates at similar extents, these modifications do not appear to hinder the comparative analysis of FFPE tissue extracts by 2-D DIGE, opening the way to its application for the differential proteomic investigation of archival tissue repositories.