Evaluation of Parameters for Confident Phosphorylation Site Localization Using an Orbitrap Fusion Tribrid Mass Spectrometer
Evaluation of Parameters for Confident Phosphorylation Site Localization Using an Orbitrap Fusion Tribrid Mass Spectrometer
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DOI：
10.1021/acs.jproteome.7b00337
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发表时间：
2017-09-01
影响因子：
4.4
通讯作者：
Eyers, Claire E.
中科院分区：
文献类型：
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作者：
Ferries, Samantha;Perkins, Simon;Eyers, Claire E.
通过质谱（MS）确定蛋白质磷酸化位点对于促进对磷酸化介导的信号传导事件的理解至关重要。然而，新仪器的发展需要MS数据采集和询问的方法进行评估和优化，高通量磷酸化蛋白质组学。在这里，我们比较和对比八种MS采集方法的新的tribrid Orbitrap融合MS平台上使用合成的磷酸肽库和复杂的磷酸肽富集的细胞裂解物。除了评估多种片段化方案（HCD，EThcD和中性丢失触发的ET（ca/hc）D）和MS/MS分析仪（轨道阱（OT）与离子阱（IT））外，我们还比较了两种常用的生物信息学平台，Andromeda与PTM评分，以及MASCOT与ptmRS，以进行可靠的磷酸肽鉴定和磷酸位点定位。我们的研究结果表明，最佳的磷酸盐鉴定是实现使用HCD碎片和高分辨率orbitrap为基础的MS/MS分析，采用MASCOT/ptmRS的数据查询。尽管EThcD对于给定PSM的置信位点定位是最佳的，但与HCD相比增加的占空比损害了鉴定的磷块岩的数量。最后，我们的数据强调，一个电荷状态依赖的碎片制度和多算法搜索策略可能是有益的自信的大规模磷酸定位。
Confident identification of sites of protein phosphorylation by mass spectrometry (MS) is essential to advance understanding of phosphorylation-mediated signaling events. However, the development of novel instrumentation requires that methods for MS data acquisition and its interrogation be evaluated and optimized for high-throughput phosphoproteomics. Here we compare and contrast eight MS acquisition methods on the novel tribrid Orbitrap Fusion MS platform using both a synthetic phosphopeptide library and a complex phosphopeptide-enriched cell lysate. In addition to evaluating multiple fragmentation regimes (HCD, EThcD, and neutral-loss triggered ET(ca/hc)D) and analyzers for MS/MS (orbitrap (OT) versus ion trap (IT)), we also compare two commonly used bioinformatics platforms, Andromeda with PTM-score, and MASCOT with ptmRS for confident phosphopeptide identification and, crucially, phosphosite localization. Our findings demonstrate that optimal phosphosite identification is achieved using HCD fragmentation and high-resolution orbitrap-based MS/MS analysis, employing MASCOT/ptmRS for data interrogation. Although EThcD is optimal for confident site localization for a given PSM, the increased duty cycle compared with HCD compromises the numbers of phosphorites identified. Finally, our data highlight that a charge-state-dependent fragmentation regime and a multiple algorithm search strategy are likely to be of benefit for confident large-scale phosphosite localization.