Zhou Ming, Meng Zhaojing, Jobson Andrew G, Pommier Yves, Veenstra Timothy D
SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, USA.
Anal Chem. 2007 Oct 15;79(20):7603-10. doi: 10.1021/ac071584r. Epub 2007 Sep 18.
A novel stable-isotope labeling approach for identification of phosphopeptides that utilizes adenosine triphosphate, in which four oxygen-16 atoms attached to the terminal phosphate group are substituted with oxygen-18 [gamma((18)O4)-ATP], has been developed. The ability to use gamma((18)O4)-ATP to monitor phosphorylation modification within various proteins was conducted by performing in vitro kinase reactions in the presence of a 1:1 mixture of gamma((18)O4)-ATP and normal isotopic abundance ATP (ATP). After tryptic digestion, the peptides were analyzed using mass spectrometry (MS). Phosphorylated peptides are easily recognized within the MS spectrum owing to the presence of doublets separated by 6.01 Da; representing versions of the peptide modified by ATP and gamma((18)O4)-ATP. Standard peptides phosphorylated using gamma((18)O4)-ATP via in vitro kinase reactions showed no exchange loss of (18)O with (16)O. The identity of these doublets as phosphorylated peptides could be readily confirmed using tandem MS. The method described here provides the first direct stable-isotope labeling method to definitely detect phosphorylation sites within proteins.
已开发出一种用于鉴定磷酸肽的新型稳定同位素标记方法，该方法利用三磷酸腺苷，其中连接到末端磷酸基团的四个氧-16原子被氧-18取代[γ((18)O4)-ATP]。通过在γ((18)O4)-ATP与正常同位素丰度的ATP（ATP）按1:1混合存在的情况下进行体外激酶反应，来检测使用γ((18)O4)-ATP监测各种蛋白质中磷酸化修饰的能力。胰蛋白酶消化后，使用质谱（MS）分析肽段。由于存在相差6.01 Da的双峰，磷酸化肽段在质谱图中很容易识别；这代表了由ATP和γ((18)O4)-ATP修饰的肽段版本。通过体外激酶反应使用γ((18)O4)-ATP磷酸化的标准肽段未显示(18)O与(16)O的交换损失。使用串联质谱可以很容易地确认这些双峰作为磷酸化肽段的身份。这里描述的方法提供了第一种直接稳定同位素标记方法，用于明确检测蛋白质中的磷酸化位点。
