ThermoFisher Scientific BRIMS, Cambridge, Massachusetts 02139, USA.
J Proteome Res. 2011 Jan 7;10(1):133-42. doi: 10.1021/pr100153j. Epub 2010 Jun 4.
The accurate diagnosis of Trisomy 21 requires invasive procedures that carry a risk of miscarriage. The current state-of-the-art maternal serum screening tests measure levels of PAPP-A, free bhCG, AFP, and uE3 in various combinations with a maximum sensitivity of 60-75% and a false positive rate of 5%. There is currently an unmet need for noninvasive screening tests with high selectivity that can detect pregnancies at risk, preferably within the first trimester. The aim of this study was to apply proteomics and mass spectrometry techniques for the discovery of new putative biomarkers for Trisomy 21 in first trimester maternal serum coupled with the immediate development of quantitative selective reaction monitoring (SRM) assays. The results of the novel workflow were 2-fold: (1) we identified a list of differentially expressed proteins in Trisomy 21 vs Normal samples, including PAPP-A, and (2) we developed a multiplexed, high-throughput SRM assay for verification of 12 new putative markers identified in the discovery experiments. To narrow down the initial large list of differentially expressed candidates resulting from the discovery experiments, we incorporated receiver operating characteristic (ROC) curve algorithms early in the data analysis process. We believe this approach provides a substantial advantage in sifting through the large and complex data typically obtained from discovery experiments. The workflow efficiently mined information derived from high-resolution LC-MS/MS discovery data for the seamless construction of rapid, targeted assays that were performed on unfractionated serum digests. The SRM assay lower limit of detection (LLOD) for the target peptides in a background of digested serum matrix was approximately 250-500 attomoles on column and the limit of accurate quantitation (LOQ) was approximately 1-5 femtomoles on column. The assay error as determined by coefficient of variation at LOQ and above ranged from 0 to 16%. The workflow developed in this study bridges the gap between proteomic biomarker discovery and translation into a clinical research environment. Specifically, for Trisomy 21, the described multiplexed SRM assay provides a vehicle for high-throughput verification of these, and potentially other, peptide candidates on larger sample cohorts.
准确诊断 21 三体需要进行有流产风险的侵入性操作。目前最先进的母体血清筛查测试通过各种组合测量 PAPP-A、游离 bhCG、AFP 和 uE3 的水平，其最大灵敏度为 60-75%，假阳性率为 5%。目前需要具有高选择性的非侵入性筛查测试来检测有风险的妊娠，最好是在孕早期。本研究旨在应用蛋白质组学和质谱技术发现与唐氏综合征相关的新的早期母体血清候选生物标志物，并立即开发定量选择性反应监测 (SRM) 分析。新工作流程的结果有两个方面：（1）我们在唐氏综合征与正常样本中鉴定出一组差异表达蛋白，包括 PAPP-A；（2）我们开发了一种多重、高通量的 SRM 分析方法，用于验证在发现实验中鉴定出的 12 个新的候选标记物。为了缩小发现实验中差异表达候选物的初始大型列表，我们在数据分析过程的早期纳入了接收者操作特征 (ROC) 曲线算法。我们相信，这种方法在筛选通常从发现实验中获得的大型和复杂数据方面具有显著优势。该工作流程有效地挖掘了来自高分辨率 LC-MS/MS 发现数据的信息，为快速、靶向分析的无缝构建提供了便利，这些分析是在未分级的血清消化物上进行的。目标肽在消化血清基质中的 SRM 分析检测限（LLOQ）约为 250-500 飞摩尔/柱，准确定量限（LOQ）约为 1-5 飞摩尔/柱。LOQ 及以上的测定误差（以变异系数表示）范围为 0-16%。本研究中开发的工作流程弥合了蛋白质组生物标志物发现与转化为临床研究环境之间的差距。具体来说，对于唐氏综合征，描述的多重 SRM 分析为在更大的样本队列中对这些肽候选物及潜在的其他候选物进行高通量验证提供了一种手段。
