Confident Phosphorylation Site Localization Using the Mascot Delta Score
Confident Phosphorylation Site Localization Using the Mascot Delta Score
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DOI：
10.1074/mcp.m110.003830
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发表时间：
2011-02-01
影响因子：
7
通讯作者：
Kuster, Bernhard
中科院分区：
文献类型：
--
作者：
Savitski, Mikhail M.;Lemeer, Simone;Kuster, Bernhard
大规模磷酸化分析越来越成为蛋白质组学研究的热点。虽然现在可以在生物系统中识别数千种磷酸化肽，但确定位点定位仍然具有挑战性。在这里，我们验证了Mascot Delta Score（MD评分）作为一种简单的方法，可以实现与已发表的Ascore（主要与Sequest结合使用）相似的磷酸盐定位灵敏度和特异性。使用液相色谱-串联MS数据的180个单独合成的磷酸化肽与精确已知的磷酸化位点的MD评分进行了评价。我们测试了广泛的常用碎片化方法的MD评分，发现它在整个过程中都适用，具有很高的统计学显著性。然而，不同的片段化技术在定位磷酸化位点的能力上存在很大差异。在1%的错误定位率，最高数量的正确分配的磷酸肽实现了更高的能量碰撞诱导解离结合的轨道阱质量分析仪，其次是非常密切的低分辨率离子阱光谱后获得的电子转移解离。这两种方法都明显优于碰撞诱导解离和多级活化后获得的低分辨率光谱。从简单的校准函数为每个片段化方法确定的分数阈值是稳定的重复分析的磷酸肽集。MD分数优于Ascore的酪氨酸磷酸化的肽，我们进一步表明，正确调用网站的能力随着两个候选网站内的肽序列的距离增加。MD分数不需要复杂的计算步骤，这使得它在实际效用方面具有吸引力。我们向社区提供所有质谱和合成肽，以便对当前和未来的本地化软件的开发进行基准测试，任何实验室都可以确定其各自分析设置的MD评分和本地化概率。Molecular & Cellular Proteomics 10：10.1074/mcp. M110.003830，1-12，2011.
Large scale phosphorylation analysis is more and more getting into focus of proteomic research. Although it is now possible to identify thousands of phosphorylated peptides in a biological system, confident site localization remains challenging. Here we validate the Mascot Delta Score (MD-score) as a simple method that achieves similar sensitivity and specificity for phosphosite localization as the published Ascore, which is mainly used in conjunction with Sequest. The MD-score was evaluated using liquid chromatography-tandem MS data of 180 individually synthesized phosphopeptides with precisely known phosphorylation sites. We tested the MD-score for a wide range of commonly available fragmentation methods and found it to be applicable throughout with high statistical significance. However, the different fragmentation techniques differ strongly in their ability to localize phosphorylation sites. At 1% false localization rate, the highest number of correctly assigned phosphopeptides was achieved by higher energy collision induced dissociation in combination with an Orbitrap mass analyzer followed very closely by low resolution ion trap spectra obtained after electron transfer dissociation. Both these methods are significantly better than low resolution spectra acquired after collision induced dissociation and multi stage activation. Score thresholds determined from simple calibration functions for each fragmentation method were stable over replicate analyses of the phosphopeptide set. The MD-score outperforms the Ascore for tyrosine phosphorylated peptides and we further show that the ability to call sites correctly increases with increasing distance of two candidate sites within a peptide sequence. The MD-score does not require complex computational steps which makes it attractive in terms of practical utility. We provide all mass spectra and the synthetic peptides to the community so that the development of present and future localization software can be benchmarked and any laboratory can determine MD-scores and localization probabilities for their individual analytical set up. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.003830, 1-12, 2011.