Optimization of TripleTOF spectral simulation and library searching for confident localization of phosphorylation sites
Optimization of TripleTOF spectral simulation and library searching for confident localization of phosphorylation sites
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DOI：
10.1371/journal.pone.0225885
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发表时间：
2019-12-02
期刊：
影响因子：
3.7
通讯作者：
Imanishi, Susumu Y.
中科院分区：
文献类型：
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作者：
Takai, Ayano;Tsubosaka, Tomoya;Imanishi, Susumu Y.
串联质谱（MS/MS）已用于分析蛋白质及其翻译后修饰。最近开发的一种数据分析方法，它模拟MS/MS光谱的磷酸化肽，并进行光谱库搜索使用SpectraST，有利于确信本地化的磷酸化位点。然而，迄今为止，其性能仅在使用Orbitrap HCD质谱仪获得的MS/MS光谱上进行了评价。在这项研究中，我们研究了这种方法是否适用于另一种类型的质谱仪，并优化了模拟和搜索条件，以实现敏感和自信的网站定位。在酶促去磷酸化之前和之后，使用TripleTOF 6600质谱仪分析合成的磷酸肽和富集的K562细胞磷酸肽。通过X！使用SimPhospho软件对串联数据库搜索进行所有可能的单磷酸化的光谱模拟。通过SpectraST对模拟光谱库进行搜索，鉴定并定位磷酸肽。尽管在先前条件下没有合成磷酸肽以1%的错误定位率定位，但是针对TripleTOF数据集的光谱模拟和搜索条件的优化实现了定位并提高了灵敏度。此外，优化的条件使K562磷酸肽的灵敏定位在1%的错误发现和定位率。这些结果表明，准确的磷酸肽模拟的TripleTOF MS/MS光谱是可能的，模拟的光谱库可以用于SpectraST搜索磷酸化位点的置信定位。
Tandem mass spectrometry (MS/MS) has been used in analysis of proteins and their post-translational modifications. A recently developed data analysis method, which simulates MS/MS spectra of phosphopeptides and performs spectral library searching using SpectraST, facilitates confident localization of phosphorylation sites. However, its performance has been evaluated only on MS/MS spectra acquired using Orbitrap HCD mass spectrometers so far. In this study, we have investigated whether this approach would be applicable to another type of mass spectrometers, and optimized the simulation and search conditions to achieve sensitive and confident site localization. Synthetic phosphopeptides and enriched K562 cell phosphopeptides were analyzed using a TripleTOF 6600 mass spectrometer before and after enzymatic dephosphorylation. Dephosphorylated peptides identified by X!Tandem database searching were subjected to spectral simulation of all possible single phosphorylations using SimPhospho software. Phosphopeptides were identified and localized by SpectraST searching against a library of the simulated spectra. Although no synthetic phosphopeptide was localized at 1% false localization rate under the previous conditions, optimization of the spectral simulation and search conditions for the TripleTOF datasets achieved the localization and improved the sensitivity. Furthermore, the optimized conditions enabled sensitive localization of K562 phosphopeptides at 1% false discovery and localization rates. These results suggest that accurate phosphopeptide simulation of TripleTOF MS/MS spectra is possible and the simulated spectral libraries can be used in SpectraST searching for confident localization of phosphorylation sites.