Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

SimPhospho 2: phosphorylation site validation in multiply phosphorylated peptides using simulated spectral libraries (#866)

Veronika Suni 1 2 , Susumu Imanishi 3 , Pekka Haapaniemi 1 , Laura Elo 1 , Garry Corthals 4
  1. Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland
  2. TUCS Turku Centre for Computer Science, Turku, Finland
  3. Faculty of Pharmacy, Meijo University, Nagoya, Japan
  4. Faculty of Science, Van ‘t Hoff Institute for Molecular Sciences, Amsterdam, The Netherlands


Site-specific identification of phosphorylation is one of the main computational challenges in phosphoproteomics. Earlier we proposed a method based on simulated phosphopeptide spectral libraries [1] that together with our software, SimPhospho [2], facilitates highly sensitive and accurate phosphosite assignments. Here, we will investigate how well multiply phosphorylated peptides can be simulated.


The principle of the simulation algorithm is as follows. Based on the spectra of dephosphorylated or nonmodified peptides, we predict spectra of phosphorylated peptides using the information about present ion peaks and their intensities as reference. Different types of ions, as well as their 2+, 3+ charged ions, and neutral losses are taken into account. Simulated spectra are collected in spectral libraries and used for identification of MS/MS spectra of phosphopeptide samples using SpectraST. For method development, hundred phosphopeptides (singly-, doubly- and triply-phosphorylated) were selected to be synthesized. These peptides were divided into three pools making sure that the phosphopeptide isoforms were separated. Analysis was done by Orbitrap HCD in Thermo Scientific Q Exactive mass spectrometer.

Results and discussion

The earlier version of SimPhospho was biased towards detection of singly phosphorylated peptides. This limitation is addressed by implementing simulation of doubly- and triply-phosphorylated peptides in SimPhospho 2. We will compare various combinations of simulation parameters in terms of False Localization Rate of phosphorylation sites we are able to obtain, when performing spectral library search of samples with known phosphorylation sites.

  1. Suni et al. Confident site localization using a simulated phosphopeptide spectral library. 2015 Journal of Proteome Research
  2. Suni et al. SimPhospho: a software tool enabling confident phosphosite assignment. 2018 Bioinformatics