Comparison of alternative MS/MS and bioinformatics approaches for confident phosphorylation site localization.
Comparison of alternative MS/MS and bioinformatics approaches for confident phosphorylation site localization.
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DOI：
10.1021/pr400402s
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发表时间：
2014-01
影响因子：
4.4
通讯作者：
Heike Wiese;K. Kuhlmann;S. Wiese;N. Stoepel;M. Pawlas;H. Meyer;C. Stephan;M. Eisenacher;F. Dreppe
中科院分区：
文献类型：
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作者：
Heike Wiese;K. Kuhlmann;S. Wiese;N. Stoepel;M. Pawlas;H. Meyer;C. Stephan;M. Eisenacher;F. Dreppe
在过去的几年里，磷酸蛋白质组学已经发展成为信号转导研究的主要工具。从那时起，已经收集了大量关于体内蛋白质磷酸化事件的信息，为更好地了解细胞信号转导的分子过程提供了宝库。然而，我们仍然面临着如何实现正确的修改站点本地化的问题。在这里，我们使用替代碎片和不同的生物信息学方法来识别和确定磷酸化位点的位置。通过多阶段活化、碰撞诱导解离和电子转移解离(ETD)对富含磷肽的组份进行分析，得到互补的磷肽鉴定。我们进一步发现，吉祥物、OMSSA和仙女座各自识别了一组不同的磷酸肽，允许位点分配的数量增加。后搜索引擎Slomo提供了可靠的磷酸化位点定位，而MaxQuant中整合的不同版本的PTM-SCORE在性能上存在差异。基于高分辨率的ETD和更高的碰撞解离(HCD)数据集，这些数据集来自由马克思等人报道的大型合成肽和磷酸肽参考库。[NAT。生物技术。2013，31(6)，557-564]，我们表明，对于HCD数据，需要仙女座/PTM得分概率为1才能提供1%的错误定位率(FLR)，而对于高分辨率ETD光谱，0.55就足够了。对HCD数据的进一步分析表明，对于磷酸酪氨酸肽和含有两个潜在磷酸化位点的磷酸肽，可以应用<；1的Ptm-Score概率截止值来确保FLR为1%。本地化概率截止点的适当调整使我们能够显著增加FLR为1%的可信站点的数量。我们的研究结果强调了有必要对不同分值的FLR进行系统评估，以报告可信的修改站点本地化。
Over the past years, phosphoproteomics has advanced to a prime tool in signaling research. Since then, an enormous amount of information about in vivo protein phosphorylation events has been collected providing a treasure trove for gaining a better understanding of the molecular processes involved in cell signaling. Yet, we still face the problem of how to achieve correct modification site localization. Here we use alternative fragmentation and different bioinformatics approaches for the identification and confident localization of phosphorylation sites. Phosphopeptide-enriched fractions were analyzed by multistage activation, collision-induced dissociation and electron transfer dissociation (ETD), yielding complementary phosphopeptide identifications. We further found that MASCOT, OMSSA and Andromeda each identified a distinct set of phosphopeptides allowing the number of site assignments to be increased. The postsearch engine SLoMo provided confident phosphorylation site localization, whereas different versions of PTM-Score integrated in MaxQuant differed in performance. Based on high-resolution ETD and higher collisional dissociation (HCD) data sets from a large synthetic peptide and phosphopeptide reference library reported by Marx et al. [Nat. Biotechnol. 2013, 31 (6), 557-564], we show that an Andromeda/PTM-Score probability of 1 is required to provide an false localization rate (FLR) of 1% for HCD data, while 0.55 is sufficient for high-resolution ETD spectra. Additional analyses of HCD data demonstrated that for phosphotyrosine peptides and phosphopeptides containing two potential phosphorylation sites, PTM-Score probability cutoff values of <1 can be applied to ensure an FLR of 1%. Proper adjustment of localization probability cutoffs allowed us to significantly increase the number of confident sites with an FLR of <1%.Our findings underscore the need for the systematic assessment of FLRs for different score values to report confident modification site localization.