Ficarro Scott B, Adelmant Guillaume, Tomar Maria N, Zhang Yi, Cheng Vincent J, Marto Jarrod A
Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute.
Anal Chem. 2009 Jun 1;81(11):4566-75. doi: 10.1021/ac9004452.
Qualitative and quantitative analysis of phosphorylation continues to be both an important and a challenging experimental paradigm in proteomics-based research. Unfortunately researchers face difficulties inherent to the optimization of complex, multivariable methods and their application to the analysis of rare and often experimentally intractable phosphorylated peptides. Here we describe a platform based on manipulation of magnetic beads in a 96-well format that facilitates rapid evaluation of experimental parameters required for enrichment of phosphopeptides. Optimized methods provided for automated enrichment and subsequent LC-MS/MS detection of over 1000 unique phosphopeptides (approximately 1% FDR) from 50 microg of cell lysates. In addition we demonstrate use of this platform for identification of phosphopeptides derived from proteins separated by SDS-PAGE and visualized near the detection limit of silver staining.
在基于蛋白质组学的研究中，磷酸化的定性和定量分析仍然是一个重要且具有挑战性的实验范式。不幸的是，研究人员面临着复杂多变量方法优化及其在分析稀有且通常实验上难以处理的磷酸化肽段时所固有的困难。在此，我们描述了一种基于在96孔板中操作磁珠的平台，该平台有助于快速评估富集磷酸肽所需的实验参数。优化后的方法可实现从50微克细胞裂解物中自动富集并随后通过液相色谱 - 串联质谱检测超过1000种独特的磷酸肽（约1%的错误发现率）。此外，我们展示了该平台用于鉴定通过SDS - PAGE分离并在银染检测限附近可视化的蛋白质衍生的磷酸肽。
