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Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

机译：shot弹枪固定，石蜡包埋样品的shot弹枪蛋白质组学分析的样品制备策略的关键比较：肝组织的见解

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Background: 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.udExperimental design: 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.udResults: 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.udConclusions: 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.
机译：背景：福尔马林固定石蜡包埋（FFPE）组织蛋白质组学的发展前景广阔，有望改善翻译研究。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shot弹枪分析的最常见工作流程，但是目前尚缺乏对不同方法的严格比较。实验设计：通过采用相同的无标记定量方法，比较了DT，FASP和ISD工作流程，每种方法均适用于FFPE肝组织的三种独立技术重复。根据方法的重现性和蛋白质/肽的分布，根据定位，分子量，pI和疏水性对数据进行了评估。 ud结果：DT显示出较低的重现性，高分子量蛋白的良好保存性，对亲水性和酸性蛋白的普遍偏向，低得多角蛋白污染，以及更高含量的非胰蛋白酶肽。相反，FASP和ISD蛋白质组中的高分子量蛋白质被消耗掉了，而疏水蛋白和膜蛋白被丰富了。 FASP可提供更高的鉴定产率，而ISD则具有更高的重现性。 ud结论：这些结果表明，多种样品制备策略可提供截然不同的蛋白质组学信息，并提出了处理FFPE样品时应考虑的典型偏见。当有足够数量的组织可用时，建议互补使用不同的方法来增加蛋白质组的覆盖范围和深度。

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