Nakanishi Tsuyoshi, Ando Eiji, Furuta Masaru, Tsunasawa Susumu, Nishimura Osamu
Life Science Laboratory, Analytical & Measuring Instruments Division, Shimadzu Corporation, 1 Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Feb 15;847(1):24-9. doi: 10.1016/j.jchromb.2006.08.024. Epub 2006 Sep 7.
We have identified tyrosine-phosphorylated proteins on membrane from A-431 human epidermoid carcinoma cells by using detection with anti-phosphotyrosine antibody followed by PMF analysis. In there, on-membrane digestion for these protein spots was carried out on microscale region using chemical inkjet technology and the resulting tryptic digests were directly analyzed by MALDI-TOF MS. Proteins identified by a database search included phosphoproteins that are known to be markedly phosphorylated on tyrosine sites after the cells are treated with epidermal growth factor (EGF). This procedure is a rapid and easily handled approach that enables both detection and identification of phosphoproteins on a single blot membrane.
我们通过使用抗磷酸酪氨酸抗体进行检测，随后进行PMF分析，鉴定了A-431人表皮样癌细胞膜上的酪氨酸磷酸化蛋白。在该实验中，使用化学喷墨技术在微观区域对这些蛋白斑点进行膜上消化，所得胰蛋白酶消化产物直接通过MALDI-TOF MS进行分析。通过数据库搜索鉴定出的蛋白质包括已知在用表皮生长因子（EGF）处理细胞后在酪氨酸位点显著磷酸化的磷酸化蛋白。该方法是一种快速且易于操作的方法，能够在单个印迹膜上同时检测和鉴定磷酸化蛋白。
