Kochin Vitaly, Imanishi Susumu Y, Eriksson John E
Turku Centre for Biotechnology, University of Turku and Abo Akademi University, BioCity, Turku, Finland.
Proteomics. 2006 Nov;6(21):5676-82. doi: 10.1002/pmic.200600457.
Tryptic phosphopeptide mapping by TLC on microcrystalline cellulose has been a convenient method to get a fast and highly reproducible overview of the number of phosphopeptides present in any given (32)P-labeled phosphoprotein. This method also provides an immediate presentation of the relative phosphorylation stoichiometry between individual phosphopeptides. However, so far, traditional tryptic phosphopeptide maps have not been useful for phosphoproteomics applications, as the S/N has been very poor, due to the large number of quenching substances and contaminants present on cellulose plates. In this study, we present a rapid and easy method for phosphopeptides identification from 2-D phosphopeptide maps (2-D-PPMs). We obtain improved sensitivity (femtomole levels) upon MALDI-TOF MS analysis of phosphopeptides extracted from 2-D-PPMs. Using this approach we could confidently characterize the major phosphorylation sites of in vivo and in vitro (32)P-labeled proteins.
通过微晶纤维素上的薄层色谱法进行胰蛋白酶磷酸肽图谱分析，一直是一种便捷的方法，可快速且高度可重复地概述任何给定的（32）P标记磷蛋白中存在的磷酸肽数量。该方法还能直接呈现各个磷酸肽之间的相对磷酸化化学计量。然而，到目前为止，传统的胰蛋白酶磷酸肽图谱对于磷酸蛋白质组学应用并无用处，因为由于纤维素板上存在大量淬灭物质和污染物，信噪比一直很差。在本研究中，我们提出了一种从二维磷酸肽图谱（2-D-PPMs）中鉴定磷酸肽的快速简便方法。对从2-D-PPMs中提取的磷酸肽进行基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析时，我们获得了更高的灵敏度（飞摩尔水平）。使用这种方法，我们能够可靠地表征体内和体外（32）P标记蛋白质的主要磷酸化位点。
