Optimization of TripleTOF spectral simulation and library searching for confident localization of phosphorylation sites.
Ayano TakaiTomoya TsubosakaYasuhiro HiranoNaoki HayakawaFumitaka TaniPekka HaapaniemiVeronika SuniSusumu Y ImanishiPublished in: PloS one (2019)
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.
Keyphrases
- ms ms
- optical coherence tomography
- tandem mass spectrometry
- data analysis
- high resolution
- ultra high performance liquid chromatography
- high performance liquid chromatography
- liquid chromatography tandem mass spectrometry
- mass spectrometry
- dual energy
- liquid chromatography
- density functional theory
- small molecule
- high throughput
- virtual reality
- single cell
- stem cells
- computed tomography
- simultaneous determination
- magnetic resonance
- rna seq
- mesenchymal stem cells
- gas chromatography
- solid phase extraction
- bone marrow
- amino acid
- adverse drug