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

Journal article


Takai, Ayano, Tsubosaka, Tomoya, Hirano, Yasuhiro, Hayakawa, Naoki, Tani, Fumitaka, Haapaniemi, Pekka, Suni, Veronika and Imanishi, Susumu Y.. (2019). Optimization of TripleTOF spectral simulation and library searching for confident localization of phosphorylation sites. PLoS ONE. 14(12), pp. 1 - 16. https://doi.org/10.1371/journal.pone.0225885
AuthorsTakai, Ayano, Tsubosaka, Tomoya, Hirano, Yasuhiro, Hayakawa, Naoki, Tani, Fumitaka, Haapaniemi, Pekka, Suni, Veronika and Imanishi, Susumu Y.
Abstract

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.

Year2019
JournalPLoS ONE
Journal citation14 (12), pp. 1 - 16
PublisherPublic Library of Science
ISSN1932-6203
Digital Object Identifier (DOI)https://doi.org/10.1371/journal.pone.0225885
Scopus EID2-s2.0-85075786223
Open accessOpen access
Page range1 - 16
Research GroupMary MacKillop Institute for Health Research
Publisher's version
License
Place of publicationUnited States of America
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