Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Mol Cell Proteomics. 2010 Feb;9(2):242-54. doi: 10.1074/mcp.M900222-MCP200. Epub 2009 Oct 26.
Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize pre-analytical and analytical variation in comparative proteomics experiments.
LC-MS/MS 平台的最佳性能对于生成高质量的蛋白质组学数据至关重要。虽然各个实验室已经开发出了质量控制样本，但目前还没有广泛可用的生物复杂性性能标准（以及相关的参考数据集），用于基准测试不同实验室之间分析复杂生物蛋白质组的平台性能。酵母 Saccharomyces cerevisiae 蛋白质组的个体制剂已被蛋白质组学领域的实验室广泛用于表征 LC-MS 平台性能。酵母蛋白质组作为性能标准具有独特的吸引力，因为它是最广泛表征的复杂生物蛋白质组，也是唯一与几个大规模研究相关联的蛋白质组，这些研究估计了所有可检测蛋白质的丰度。在这项研究中，我们描述了大规模生产酵母性能标准的标准操作程序，并通过美国国家标准与技术研究院（NIST）向社区提供样品，该研究院正在开发酵母蛋白质组作为认证参考物质，以满足社区的长期需求。我们使用一系列描述 LC-MS 性能的指标，提供了一个参考数据集，展示了常用离子阱仪器平台在专家实验室中的典型性能；结果为实验室提供了基准测试自己的性能、改进当前方法和评估新技术的基础。此外，我们还展示了如何使用酵母参考品（添加了人类蛋白质）来基准化蛋白质组学平台在复杂基质中不同浓度水平下检测差异表达蛋白质的能力，从而提供了一种评估和最小化比较蛋白质组学实验中预分析和分析变异性的指标。
