Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue.
Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue.
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DOI：
10.1186/1559-0275-11-28
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发表时间：
2014
影响因子：
3.8
通讯作者：
Addis MF
中科院分区：
文献类型：
--
作者：
Tanca A;Abbondio M;Pisanu S;Pagnozzi D;Uzzau S;Addis MF
福尔马林固定石蜡包埋 (FFPE) 组织蛋白质组学领域的不断发展为改善转化研究带来了希望。直接组织胰蛋白酶消化 (DT) 和蛋白质提取，然后进行溶液消化 (ISD) 或过滤辅助样品制备 (FASP) 是 FFPE 样品鸟枪法分析最常见的工作流程，但目前缺乏对不同方法的关键比较。通过采用相同的无标记定量方法，对应用于 FFPE 肝脏组织的每种方法进行三次独立的技术重复，对 DT、FASP 和 ISD 工作流程进行比较。根据定位、MW、pI 和疏水性，从方法重现性和蛋白质/肽分布方面评估数据。 DT 显示出较低的重现性、高分子量蛋白质的良好保存、对亲水性和酸性蛋白质的普遍偏向、角蛋白污染要低得多，以及非胰蛋白酶肽的丰度较高。相反，FASP 和 ISD 蛋白质组中高分子量蛋白质被耗尽，而疏水性和膜蛋白被富集； FASP 提供了更高的鉴定率，而 ISD 则表现出更高的再现性。这些结果强调，不同的样品制备策略提供了显着不同的蛋白质组信息，并呈现出处理 FFPE 样品时应考虑到的典型偏差。当有足够量的组织可用时，建议补充使用不同的方法以增加蛋白质组的覆盖范围和深度。
The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking. DT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity. DT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility. These results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.
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