Quantitative Comparison of Deparaffinization, Rehydration, and Extraction Methods for FFPE Tissue Proteomics and Phosphoproteomics,Analytical Chemistry

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Quantitative Comparison of Deparaffinization, Rehydration, and Extraction Methods for FFPE Tissue Proteomics and Phosphoproteomics
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-08-05 , DOI: 10.1021/acs.analchem.3c04479
Erin M Humphries ₁ , Clare Loudon ₁ , George E Craft ₁ , Peter G Hains ₁ , Phillip J Robinson ₁

Affiliation

Formalin-fixed paraffin-embedded (FFPE) tissues are suitable for proteomic and phosphoproteomic biomarker studies by data-independent acquisition mass spectrometry. The choice of the sample preparation method influences the number, intensity, and reproducibility of identifications. By comparing four deparaffinization and rehydration methods, including heptane, histolene, SubX, and xylene, we found that heptane and methanol produced the lowest coefficients of variation (CVs). Using this, five extraction methods from the literature were modified and evaluated for their performance using kidney, leg muscle, lung, and testicular rat organs. All methods performed well, except for SP3 due to insufficient tissue lysis. Heat n’ Beat was the fastest and most reproducible method with the highest digestion efficiency and lowest CVs. S-Trap produced the highest peptide yield, while TFE produced the best phosphopeptide enrichment efficiency. The quantitation of FFPE-derived peptides remains an ongoing challenge with bias in UV and fluorescence assays across methods, most notably in SPEED. Functional enrichment analysis demonstrated that each method favored extracting some gene ontology cellular components over others including chromosome, cytoplasmic, cytoskeleton, endoplasmic reticulum, membrane, mitochondrion, and nucleoplasm protein groups. The outcome is a set of recommendations for choosing the most appropriate method for different settings.

中文翻译：

FFPE 组织蛋白质组学和磷酸化蛋白质组学脱蜡、复水和提取方法的定量比较

福尔马林固定石蜡包埋 (FFPE) 组织适用于通过数据独立采集质谱法进行蛋白质组和磷酸化蛋白质组生物标志物研究。样品制备方法的选择会影响鉴定的数量、强度和重现性。通过比较四种脱蜡和再水化方法，包括庚烷、histolene、SubX 和二甲苯，我们发现庚烷和甲醇产生最低的变异系数 (CV)。利用这一点，对文献中的五种提取方法进行了修改，并使用肾脏、腿部肌肉、肺和睾丸大鼠器官评估了它们的性能。除 SP3 因组织裂解不充分外，所有方法均表现良好。 Heat n' Beat 是最快、最具重复性的方法，具有最高的消化效率和最低的 CV。 S-Trap 产生的肽产量最高，而 TFE 产生的磷酸肽富集效率最好。 FFPE 衍生肽的定量仍然是一个持续的挑战，不同方法（尤其是 SPEED）中的 UV 和荧光测定存在偏差。功能富集分析表明，每种方法都比其他方法更有利于提取一些基因本体细胞成分，包括染色体、细胞质、细胞骨架、内质网、膜、线粒体和核质蛋白组。结果是针对不同设置选择最合适方法的一组建议。

更新日期：2024-08-05

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