Optimization of TripleTOF spectral simulation and library searching for confident localization of phosphorylation sites

Autoři: Ayano Takai aff001;  Tomoya Tsubosaka aff001;  Yasuhiro Hirano aff001;  Naoki Hayakawa aff001;  Fumitaka Tani aff001;  Pekka Haapaniemi aff002;  Veronika Suni aff002;  Susumu Y. Imanishi aff001
Působiště autorů: Faculty of Pharmacy, Meijo University, Nagoya, Japan aff001;  Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland aff002;  Turku Centre for Computer Science, Turku, Finland aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225885


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.

Klíčová slova:

Data acquisition – Database searching – Phosphorylation – Sequence databases – Serine – Synthetic peptides – Peptide libraries – Mass spectrometers


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