Kennedy Jacob J, Whiteaker Jeffrey R, Schoenherr Regine M, Yan Ping, Allison Kimberly, Shipley Melissa, Lerch Melissa, Hoofnagle Andrew N, Baird Geoffrey Stuart, Paulovich Amanda G
Clinical Research Division, Fred Hutchinson Cancer Research Center , Seattle, Washington 98109, United States.
Department of Pathology, Stanford University , Stanford, California 94305 United States.
J Proteome Res. 2016 Aug 5;15(8):2717-28. doi: 10.1021/acs.jproteome.6b00245. Epub 2016 Jul 27.
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.
尽管开发伴随诊断在临床、经济和监管方面都具有紧迫性，但成功转化为临床应用的新生物标志物却寥寥无几，部分原因是蛋白质检测技术不足以支持对福尔马林固定石蜡包埋（FFPE）组织中的数百种候选生物标志物进行大规模检测。虽然已证明使用靶向多反应监测质谱（MRM-MS）对FFPE组织进行定量分析的可行性，但尚未针对大量分析物的稳健定量对方案进行系统优化，也未评估肽免疫MRM的性能。为了填补这一空白，我们采用了一种测试组合方法，结合MRM-MS并添加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）组合的性能。确定了一种基于RapiGest缓冲液提取和尿素消化的过程，能够从FFPE组织和冷冻组织中获得相似的定量结果。使用针对FFPE组织基于MRM分析的优化方案，中位精密度为11.4%（在249种分析物中）。对于直接MRM分析（R(2)=0.94）和免疫MRM（R(2)=0.89），在匹配的FFPE组织和冷冻组织上进行的测量之间具有极好的相关性。优化后的过程能够在存档组织标本中实现高度可重复、多重、标准化的定量MRM。
