Molina Henrik, Horn David M, Tang Ning, Mathivanan Suresh, Pandey Akhilesh
McKusick-Nathans Institute for Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2199-204. doi: 10.1073/pnas.0611217104. Epub 2007 Feb 7.
Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique that provides a more comprehensive coverage of peptide sequences and posttranslational modifications. Here, we evaluated the use of ETD for a global phosphoproteome analysis. In all, we identified a total of 1,435 phosphorylation sites from human embryonic kidney 293T cells, of which 1,141 ( approximately 80%) were not previously described. A detailed comparison of ETD and collision-induced dissociation (CID) modes showed that ETD identified 60% more phosphopeptides than CID, with an average of 40% more fragment ions that facilitated localization of phosphorylation sites. Although our data indicate that ETD is superior to CID for phosphorylation analysis, the two methods can be effectively combined in alternating ETD and CID modes for a more comprehensive analysis. Combining ETD and CID, from this single study, we were able to identify 80% of the known phosphorylation sites in >1,000 phosphorylated peptides analyzed. A hierarchical clustering of the identified phosphorylation sites allowed us to discover 15 phosphorylation motifs that have not been reported previously. Overall, ETD is an excellent method for localization of phosphorylation sites and should be an integral component of any strategy for comprehensive phosphorylation analysis.
电子转移解离（ETD）是一种最近引入的质谱技术，它能更全面地覆盖肽序列和翻译后修饰。在此，我们评估了ETD在全局磷酸化蛋白质组分析中的应用。我们总共从人胚肾293T细胞中鉴定出1435个磷酸化位点，其中1141个（约80%）此前未被描述。对ETD和碰撞诱导解离（CID）模式的详细比较表明，ETD鉴定出的磷酸肽比CID多60%，促进磷酸化位点定位的碎片离子平均多40%。尽管我们的数据表明ETD在磷酸化分析方面优于CID，但这两种方法可以通过交替使用ETD和CID模式有效地结合起来进行更全面的分析。结合ETD和CID，从这项单一研究中，我们能够在分析的1000多个磷酸化肽中鉴定出80%的已知磷酸化位点。对鉴定出的磷酸化位点进行层次聚类使我们发现了15个此前未报道过的磷酸化基序。总体而言，ETD是一种用于磷酸化位点定位的优秀方法，应该是任何全面磷酸化分析策略中不可或缺的组成部分。
