Comparative evaluation of two methods for LC-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues.
Comparative evaluation of two methods for LC-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues.
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DOI：
10.1016/j.jprot.2021.104117
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发表时间：
2021-03-20
影响因子：
3.3
通讯作者：
Dwork AJ
中科院分区：
文献类型：
--
作者：
Davalieva K;Kiprijanovska S;Dimovski A;Rosoklija G;Dwork AJ
福尔马林固定，石蜡包埋（FFPE）样品的蛋白质组学在过去的二十年中取得了显着进展，但有许多协议和很少的研究直接比较它们。对于鸟枪法蛋白质组学分析的最有效的方案没有共识。我们使用两种无标记数据独立LC-MS/MS采集，比较了储存7年的FFPE前列腺组织和镜像新鲜冷冻样本的RapiGest和过滤辅助样品制备（FASP）的溶液内消化。RapiGest比FASP鉴定了更多的蛋白质，新鲜组织和FFPE组织中的蛋白质数量几乎相同，重叠率为69%，高分子量蛋白质保存良好，等电点无偏倚，技术重现性更好。另一方面，与新鲜组织相比，FASP在FFPE中产生的蛋白质鉴定少20%，具有64-69%的重叠，> 70 kDa的蛋白质的耗尽，在酸性和中性范围内的效率较低，并且技术再现性较低。两种方案均显示出高度相似的亚细胞区室分布、从FFPE和新鲜组织中提取的独特肽的高度相似的百分比以及新鲜和FFPE蛋白质的绝对定量值之间的高度正相关性。总之，FFPE组织的RapiGest提取物提供了与新鲜冷冻蛋白质组非常相似的蛋白质组，在生物标志物和定量研究中应优于FASP。
The proteomics of formalin-fixed, paraffin-embedded (FFPE) samples has advanced significantly during the last two decades, but there are many protocols and few studies comparing them directly. There is no consensus on the most effective protocol for shotgun proteomic analysis. We compared the in-solution digestion with RapiGest and Filter Aided Sample Preparation (FASP) of FFPE prostate tissues stored 7 years and mirroring fresh frozen samples, using two label-free data-independent LC-MS/MS acquisitions. RapiGest identified more proteins than FASP, with almost identical numbers of proteins from fresh and FFPE tissues and 69% overlap, good preservation of high-MW proteins, no bias regarding isoelectric point, and greater technical reproducibility. On the other hand, FASP yielded 20% fewer protein identifications in FFPE than in fresh tissue, with 64–69% overlap, depletion of proteins >70kDa, lower efficiency in acidic and neutral range, and lower technical reproducibility. Both protocols showed highly similar subcellular compartments distribution, highly similar percentages of extracted unique peptides from FFPE and fresh tissues and high positive correlation between the absolute quantitation values of fresh and FFPE proteins. In conclusion, RapiGest extraction of FFPE tissues delivers a proteome that closely resembles the fresh frozen proteome and should be preferred over FASP in biomarker and quantification studies.
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影响因子：
7
作者：
Hood, BL;Darfler, MM;Krizman, DB
通讯作者：
Krizman, DB
影响因子：
3
作者：
Davalieva, Katarina;Kiprijanovska, Sanja;Polenakovic, Momir
通讯作者：
Polenakovic, Momir
影响因子：
4.4
作者：
Kennedy, Jacob J.;Whiteaker, Jeffrey R.;Paulovich, Amanda G.
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Paulovich, Amanda G.
影响因子：
3.2
作者：
Ikeda, K;Monden, T;Monden, M
通讯作者：
Monden, M
影响因子：
7.3
作者：
Nirmalan, Niroshini J.;Harnden, Patricia;Banks, Rosamonde E.
通讯作者：
Banks, Rosamonde E.