Optimized Protocol for Quantitative Multiple Reaction Monitoring Based Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissues
Optimized Protocol for Quantitative Multiple Reaction Monitoring Based Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissues
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DOI：
10.1021/acs.jproteome.6b00245
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发表时间：
2016-08-01
影响因子：
4.4
通讯作者：
Paulovich, Amanda G.
中科院分区：
文献类型：
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作者：
Kennedy, Jacob J.;Whiteaker, Jeffrey R.;Paulovich, Amanda G.
尽管开发伴随诊断在临床、经济和监管方面都势在必行，但很少有新的生物标志物成功转化为临床应用，部分原因是蛋白质测定技术不足以支持在福尔马林固定石蜡包埋 (FFPE) 组织中对数百种候选生物标志物进行大规模测试。尽管使用靶向多反应监测质谱 (MRM-MS) 进行 FFPE 组织定量分析的可行性已得到证实，但尚未系统优化方案以实现大量分析物的稳健定量，也未评估肽免疫 MRM 的性能。为了解决这一差距，我们使用了与 MRM-MS 结合的测试电池方法，并添加了稳定同位素标记的标准肽（针对 512 种分析物），以定量评估三种提取方案与三种胰蛋白酶消化方案（即九个过程）相结合的性能。确定了基于 RapiGest 缓冲液提取和尿素消化的工艺，可以从 FFPE 和冷冻组织中获得类似的定量结果。使用优化的方案对 FFPE 组织进行基于 MRM 的分析，中位精度为 11.4%（涉及 249 种分析物）。对于直接 MRM 分析 (R-2 = 0.94) 和免疫 MRM 分析 (R-2 = 0.89)，在匹配的 FFPE 和冷冻组织上进行的测量之间存在极好的相关性。优化的流程可在档案组织样本中实现高度可重复、多重、可标准化、定量的 MRM。
Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R-2 = 0.94) and immuno-MRM (R-2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens.