4 HANÁKOVÁ, POTĚŠIL, BERNATÍK, ČERVENKA, RÁDSETOULAL, BRYJA, AND ZDRÁHAL Figure 1: Comparison of phosphorylation sites of hDvl3 induced by CK1ε and NEK2. Experimentally determined phospho- rylation sites by CK1ε and NEK2 are indicated by green background color. Phosphorylation sites identified only by NEK2 are indicated by red background color. analyzed by liquid chromatography coupled with mass spectrometry (LC-MS/MS) for protein identification (ID run). The rest of the peptide mixture was used for phospho- peptide analysis. MS PhosphoMix 1, 2, 3 Light (Sigma Aldrich) was added to the samples before the phos- phopeptide enrichment step in a concentration of 0.1 pmol. Phosphopeptides were enriched using a Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the protocol of the manufacturer and eluted into an autosampler vial. The solution was con- centrated under a vacuum to a volume of less than 5 µl, dissolved in water and 0.6 µl of 5% FA was used to obtain 15 µl of the peptide solution before LC-MS/MS analysis. 2.3 Mass spectrometry LC-MS/MS analyses of the peptide mixture were con- ducted using a RSLCnano system connected to a Orbitrap Elite hybrid mass spectrometer (Thermo Fisher Scien- tific) with a ABIRD (Active Background Ion Reduction Device; ESI Source Solutions) and a Digital PicoView DPV550 (New Objective) ion source (tip rinsing by 50% ACN with 0.1% FA) installed. Prior to LC separation, tryptic digests were concentrated online and desalted us- ing a trapping column (100 µm × 30 mm) filled with 3.5-µm of XBridge BEH 130Å C18 sorbent (Waters). Af- ter the trapping column was washed with 0.1% FA, the peptides were eluted (flow rate of 300 nl/min) from the trapping column onto a Acclaim PepMap100 C18 col- umn (3 µm particles, 75 µm × 500 mm; Thermo Fisher Scientific) along a 65 min-long gradient. Mobile phase A (0.1% FA in water) and mobile phase B (0.1% FA in 80% ACN) were used in both cases. The gradient elution started at 1% of mobile phase B and increased from 1% to 56% during the first 50 mins (30% in the 35th and 56% in the 50th min), then increased linearly to 80% of mobile phase B over the following 5 mins and remained at this state for the next 10 mins. Equilibration of the trapping column and the anlytical column was conducted prior to injection of the sample into the sample loop. The outlet of the analytical column was directly connected to the Dig- ital PicoView DPV550 ion source. MS data were acquired in a data-dependent strategy by selecting the top 6 precursors based on precursor abun- dance in the survey scan (350-2000 m/z). The resolution of the survey scan was 60,000 (at 400 m/z) with a tar- get value of 1×106 ions, one microscan and a maximum injection time of 200 ms. High resolution (resolution of 15,000 at 400 m/z) higher energy collisional dissociation (HCD) MS/MS spectra were acquired with a target value of 50,000. The normalized collision energy was 32 % for HCD spectra. The maximum injection time for MS/MS was 500 ms. Dynamic exclusion was enabled for 45 s af- ter the acquisition of one MS/MS spectra and early ex- piration was disabled. The isolation window for MS/MS fragmentation was set to 2 m/z. The analysis of the mass spectrometric RAW data files was carried out using the Proteome Discov- erer software (Thermo Fisher Scientific; version 1.4) with utilization of the in-house Mascot (Matrixscience; version 2.4.1) search engine. MS/MS ion searches were conducted against an in-house database con- taining the expected protein of interest with addi- tional sequences from the cRAP (common Repository of Adventitious Proteins) database (downloaded from http://www.thegpm.org/crap/). Mass tolerance for pep- tides and MS/MS fragments were 7 ppm and 0.03 Da, re- spectively. Oxidation of methionine, deamidation (N, Q) Hungarian Journal of Industry and Chemistry SEMIQUANTITATIVE ANALYSIS OF DVL3 PHOSPHORYLATIONS 5 Figure 2: Semiquantitative analysis of phosphorylation site serine 204 (S204) on peptide FSSpSTEQSSASR induced by CK1ε and NEK2. Precursor and selected fragment traces of corresponding hDvl3 phosphopeptides are shown for CK1ε and NEK2 (in Skyline). The highest signal intensity was detected in the case of NEK2. and phosphorylation (S, T, Y) as optional modifications, carbamidomethylation of C as a fixed modification and three miss cleavages of enzymes were set for all searches. The phosphoRS feature was used for phosphorylation site localization. Quantitative information was assessed and manually validated in Skyline software (Skyline-daily 3.1.1.8884). 3. Results and Analysis 3.1 Identification of phosphorylation sites Phosphorylation is important for protein function and regulation. The phosphorylation status of human Dvl3 in- duced by eight individual Ser/Thr kinases that were previ- ously reported or identified by an unbiased MS screen for Dvl-associated kinases was analysed. Dvl3 contains 131 serines/threonines, which can be potentially phosphory- lated. In total, 88 Ser/Thr phosphorylation sites and one tyrosine phosphorylation site in Dvl3 were identified. 3.2 Phosphorylations induced by CK1ε and NEK2 Based on our experiment, a phosphorylation “map” of the Dvl protein was created that described the complex phos- phorylation “fingerprint” for each kinase tested. Eight of the kinases used to induce phosphorylation include CK1ε and NEK2. Fig. 1 shows a qualitative comparison of the identified phosphorylation sites using these two kinases. In the case of CK1ε induction, 77 phosphorylation sites were identified, and in the case of NEK2, 87 phosphory- lation sites were determined from a total of 131 possible Ser/Thr phosphorylation sites in the Dvl3 protein. Next in terms of qualitative characterization, a semi- quantitative comparison with regard to the occupancy of phosphorylation sites induced by individual kinases was conducted. The Skyline software was used for this evalu- ation. The individual phosphorylated peptides were com- pared based on their peak areas. A comparison of a se- lected peptide phosphorylated in the position of S204 by CK1ε and NEK2 is shown in Fig. 2. The peak area was determined for CK1ε as 7.60e6 and for NEK2 as 1.07e9. Subsequently, double normalization of the data was per- formed using a set of phosphopeptide standards (added to the sample prior to the phospho-enrichment step) and by unphosphorylated peptides identified in the identifica- tion run. The resulting areas (CK1ε: 1.23e7 and NEK2: 1.10e9) were compared with each other. 4. Discussion Our study focused on the determination of the phospho- rylation sites of Dvl3 by MS induced by eight kinases. 88 Ser/Thr phosphorylations from a total of 131 sites and 1 tyrosine phosphorylation were identified which can be potentially phosphorylated. CK1ε-induced phosphorylation was identified at 77 unique sites and 10 more phosphorylation sites were in- duced by NEK2. Previous studies in various experimental systems identified several phosphorylation sites spread throughout the structure of the protein Dvl3 [3, 4]. Our data clearly demonstrate that the phosphorylation of the protein Dvl3 is extensive and the number of phosphory- lated sites exceeds 60. 5. Conclusion An approach based on the SDS-PAGE separation of Dvl3 immunoprecipitates, TiO2 phospho-enrichment followed by LC-MS/MS analysis and data processing using Sky- line software was utilized for the evaluation of semiquan- titative differences in the phosphorylation level of hDvl3 at particular sites within the set of eight selected kinases. 46(1) pp. 3-6 (2018) 6 HANÁKOVÁ, POTĚŠIL, BERNATÍK, ČERVENKA, RÁDSETOULAL, BRYJA, AND ZDRÁHAL Differences were observed in terms of the phosphory- lation profiles induced by individual kinases, as indicated in Fig. 1. Based on our results, a “comprehensive map” of the phosphorylations of human Dvl3 will be created. Acknowledgement This work was carried out with the support of the project CEITEC 2020 (LQ1601) funded by the Ministry of Ed- ucation, Youth and Sports (MEYS) of the Czech Re- public under the National Sustainability Programme II. The Czech Infrastructurefor Integrative Structural Biol- ogy (CIISB) research infrastructure project LM2015043 funded by MEYS is gratefully acknowledged for finan- cially supporting our LC-MS/MS measurements at the Proteomics Core Facility. The support from the Czech Science Foundation project no. 15-21789S is also grate- fully acknowledged. REFERENCES [1] Kersten, B., Agrawal, G. K., Iwahashi, H., Rak- wal, R.: Plant phosphoproteomics: A long road ahead, Proteomics, 2006 6(20), 5517–5528 DOI: 10.1002/pmic.200600232 [2] Bernatík, O., Šedová, K., Schille, C., Ganji, S. R., Červenka, I., Trantírek, L., Schambony, A., Zdráhal, Z., Bryja, V.: Functional analysis of Dishevelled- 3 phosphorylation identifies distinct mechanisms driven by casein kinase 1 epsilon and frizzled5, J. Biol. Chem., 2014 34(289), 23520-23533 DOI: 10.1074/jbc.M114.590638 [3] Yanfeng, W. A., Berhane, H., Mola, M., Singh, J., Jenny, A., Mlodzik, M.: Functional dissection of phosphorylation of Disheveled in Drosophila, Dev. Biol., 2011 360, 132–142 DOI: 10.1016/j.ydbio.2011.09.017 [4] Klimowski, L. K., Garcia, B. A., Shabanowitz, J., Hunt, D. F., Virshup, D. M.: Site-specific casein ki- nase 1�-dependent phosphorylation of Dishevelled modulates β-catenin signaling, FEBS J., 2006 273, 4594–4602 DOI: 10.1111/j.1742-4658.2006.05462.x Hungarian Journal of Industry and Chemistry Introduction Experimental Cell culture and transfection Gel electrophoresis, protein digestion and phosphopeptide enrichment Mass spectrometry Results and Analysis Identification of phosphorylation sites Phosphorylations induced by CK1 and NEK2 Discussion Conclusion