整合聚精胺酸親和萃取，膠體電泳和質譜方法於磷酸化蛋白質體學的研究

不管是在磷酸化蛋白質的找尋或是磷酸化的位置，磷酸化蛋白質萃取的過程往往都是繁複但卻是不可避免的。所以我們在此提供一個方法，利用奈米鑽石修飾上聚精胺酸，從蛋白質混合物或是細胞萃取液裡頭萃取出磷酸化蛋白質。萃取出的蛋白質和奈米鑽石粒子可以直接加進膠體電泳中，經過膠體內消化作用，再利用質譜方法分析而得到蛋白質磷酸化的位置。在經由不斷的嘗試之下我們利用酪蛋白找出最佳反應溶液的化合物組成(pH 值3 、1.5%CHAPS 以及0.4M 氯化鈉)，且酪蛋白的回收率超過90％。而此方法的高專一性可以藉由10 倍的非磷酸化物質存在環境下得到驗證。同時，經由Pro-Q 的磷酸化蛋白質染劑的證明，對於真實樣品子宮頸癌細胞的萃取也能作用。以上實驗結果可以確認，此方法不但有效，而且未來同樣能應用在磷酸化蛋白質學的研究上。

關鍵字

聚精胺酸；磷酸化蛋白質；膠體電泳；子宮頸癌細胞

並列摘要

Sample prefractionation is often a tedious but indispensable procedure for the identification of phosphoproteins and sites of phosphorylation. Here we described an integrated approach to selectively enrich phosphoproteins from protein mixtures and cell lysates using poly-arginine coated nanodiamonds (PA-NDs) as extraction platforms. Affinity captured proteins on NDs were directly subjected to1-D PAGE for fractionation and sites of phosphorylation were further identified by affinity purification mass spectrometry after in-gel digestion. Detailed analyses of the pH and ionic strength for the purification of β-casein from a standard protein mixture enabled us to determine the optimal conditions (pH 3 and 0.4 m NaCl) and the recovery of β-casein was more than 90%. The high specificity was validated by isolating phosphoproteins from a standard protein mixture with a 1:10 β-casein/myoglobin molar ratio. The integrated approach was also applied to enrich phosphorylated proteins for HeLa cell lysates and revealed most of the extracted proteins are phosphorylated by Pro-Q diamond phosphoprotein staining method. These results demonstrate the efficacy of the new approach for phosphoprotein characterization and expected to be potentially useful in phosphoproteome research.