Abbatiello Susan E, Schilling Birgit, Mani D R, Zimmerman Lisa J, Hall Steven C, MacLean Brendan, Albertolle Matthew, Allen Simon, Burgess Michael, Cusack Michael P, Gosh Mousumi, Hedrick Victoria, Held Jason M, Inerowicz H Dorota, Jackson Angela, Keshishian Hasmik, Kinsinger Christopher R, Lyssand John, Makowski Lee, Mesri Mehdi, Rodriguez Henry, Rudnick Paul, Sadowski Pawel, Sedransk Nell, Shaddox Kent, Skates Stephen J, Kuhn Eric, Smith Derek, Whiteaker Jeffery R, Whitwell Corbin, Zhang Shucha, Borchers Christoph H, Fisher Susan J, Gibson Bradford W, Liebler Daniel C, MacCoss Michael J, Neubert Thomas A, Paulovich Amanda G, Regnier Fred E, Tempst Paul, Carr Steven A
From the Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142;
Buck Institute for Research on Aging, Novato, California 94945;
Mol Cell Proteomics. 2015 Sep;14(9):2357-74. doi: 10.1074/mcp.M114.047050. Epub 2015 Feb 18.
There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.
在生物学和临床医学领域，对于在临床和生物样本中以高灵敏度、特异性、重现性和可重复性稳健且可靠地测量数十至数百种肽和蛋白质的需求日益增长。此前，我们证明了采用同位素稀释的液相色谱-多反应监测-质谱联用（LC-MRM-MS）技术在定量测量人血浆中少量相对丰富的蛋白质方面具有合适的性能，并且由此得到的检测方法能够在不同实验室间转移，同时保持高重现性和定量精度。在此，我们显著扩展了早期的工作，证明了11个实验室使用14台液相色谱-质谱联用（LC-MS）系统能够开发、确定分析性能指标，并应用针对源自27种癌症相关蛋白和7种对照蛋白的125种肽的高度多重化MRM-MS检测方法，以精确且可重复地测量人血浆中的分析物。为确保始终生成高质量数据，我们在实验设计中纳入了系统适用性方案（SSP）。该SSP能够在检测方法开发和实施过程中实时监测LC-MRM-MS的性能，便于早期检测和纠正色谱及仪器问题。通过在分析前对14种丰富的血浆蛋白进行一步免疫亲和去除，实现了血浆中蛋白质低至亚纳克/毫升的灵敏度。实验室内和实验室间的中位重现性均小于20%，这对于大多数生物学研究和候选蛋白质生物标志物验证而言已足够。使用蛋白质的重同位素标记版本作为内标评估了肽的消化回收率并提高了定量准确性。通过使用高度多重化检测方法，参与研究的实验室能够精确且可重复地确定用于模拟患者样本实验室间临床研究的盲法样本中一系列分析物的水平。我们的研究进一步证实，采用稳定同位素稀释的LC-MRM-MS技术，在适当关注分析验证和采取适当质量控制措施的情况下，能够对诸如血浆等复杂生物基质中的蛋白质和肽进行灵敏、特异、可重复和定量的测量。
