Rogers Lindsay D, Fang Yuan, Foster Leonard J
Centre for High-Throughput Biology and Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada.
Mol Biosyst. 2010 May;6(5):822-9. doi: 10.1039/b915986j. Epub 2010 Feb 3.
Recently, the field of phosphoproteomics has progressed to the point where thousands of protein phosphorylations can be analyzed simultaneously and used to address significant biological questions. However, several challenges still exist in current LC-MS/MS-based phosphoproteomics methods. Among these are the increased dynamic range of phosphoproteomics samples (due to low stoichiometry of most protein phosphorylations), insufficient inhibition of phosphatase activity, and neutral losses which occur during phosphopeptide fragmentation by MS(n). Here we present an improved method, free of conventional phosphatase inhibitors, for sample treatment to minimize phosphatase activity and improve the efficiency of phosphopeptide enrichment. We also present a solution-based IEF method for phosphopeptide fractionation and explore the utility of various fragmentation methods for identifying phosphopeptides and localizing phosphorylation sites.
最近，磷酸化蛋白质组学领域已经发展到可以同时分析数千种蛋白质磷酸化，并用于解决重要生物学问题的阶段。然而，当前基于液相色谱-串联质谱（LC-MS/MS）的磷酸化蛋白质组学方法仍然存在一些挑战。其中包括磷酸化蛋白质组学样品动态范围的增加（由于大多数蛋白质磷酸化的化学计量较低）、磷酸酶活性抑制不足以及在串联质谱（MS(n)）对磷酸肽进行碎片化过程中发生的中性丢失。在此，我们提出一种改进的方法，无需使用传统的磷酸酶抑制剂进行样品处理，以最大限度地降低磷酸酶活性并提高磷酸肽富集效率。我们还提出了一种基于溶液的等电聚焦（IEF）方法用于磷酸肽分级分离，并探索了各种碎片化方法在鉴定磷酸肽和定位磷酸化位点方面的实用性。
