Supporting Information Global analysis of phosphoproteome dynamics in embryonic development of zebrafish (Danio rerio) Oh Kwang Kwon1, Sun Ju Kim1, You Mie Lee1, Young-Hoon Lee2, Young-Seuk Bae2, Jin Young Kim3, Xiaojun Peng4, Zhongyi Cheng5, Yingming Zhao6, Sangkyu Lee1,* 1 College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, South Korea 2 School of Life Sciences, KNU Creative BioResearch Group (BK21 plus program), Kyungpook National University, Daegu 41566, Korea 3 Mass Spectrometry Research Center, Korea Basic Science Institute, Ochang, Chungbuk 28115, Republic of Korea 4 Jingjie PTM Biolabs (Hangzhou) Co. Ltd, Hangzhou, China 310018 5 Advanced Institute of Translational Medicine, Tongji University, Shanghai 200092, China 6 Ben May Department for Cancer Research, The University of Chicago, Chicago, IL 60637, USA *correspondence: Sangkyu Lee, Ph.D., Professor, College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu 702-701, Republic of Korea. Tel.: +82-53-950 8571; Fax: +82-53-950-8557; E-mail: sangkyu@knu.ac.kr. 1 Supporting Methods 1. Materials The phosphopeptide enrichment TiO2 kits, GL-Tip SDB tip, and GL-Tip GC tip, were purchased from GL Sciences. Modified sequencing-grade trypsin was purchased from Promega. Trifluoroacetic acid, trichloroacetic acid, formic acid, glycolic acid, dithiothreitol, iodoacetamide, and cysteine were purchased from Sigma-Aldrich. Antibody against pSer was obtained from abcam®. Antibodies against pThr and pTye were purchased from Cell Signaling Technology®. Complete RIPA buffer, C18 Tips, and the BCA protein assay kit, and ECL Plus western blotting substrates were purchased from Thermo Scientific. 18 O water (purified 97%) was purchased from Cambridge Isotope Laboratories, and ammonium bicarbonate was obtained from Daejung. Polyvinylidene difluoride membranes were purchased from Roche. Protease inhibitor cocktail and phosphatase inhibitor cocktail were from Merck Millipore. Acetone, high-performance liquid chromatography (HPLC)-grade acetonitrile, and HPLC-grade water were purchased from Burdick and Jackson. Water was purified using the Milli Q system (Millipore). 2. CK2 activity assay The standard assay for phosphotransferase activity of CK2 was conducted in a 30-µL reaction mixture containing 20 mM Tris-HCl (pH 7.5), 120 mM KCl, 10 mM MgCl2, and 100 µM [32P]ATP in the presence of 1 mM synthetic peptide substrate (RRREEETEEE) at 30°C. Cell lysates were added to initiate the reaction and incubated for 15 min. The reaction was stopped by the addition of trichloroacetic acid to a final concentration of 10%. The mixture was then centrifuged, after which 10 µL of the supernatant was applied to P-81 paper. The paper was washed in 100 mM phosphoric acid, and radioactivity was measured by scintillation counting. 3. Immunoblotting and Immunoprecipitation For immunoprecipitation, the protein extracts (500 g of total protein) were then incubated overnight with the phosphoserine antibody (abcam) at 4oC followed by incubation for 4 hour at 4 oC with a 50 % slurry of protein G 2 magnetic beads. After washing immunoprecipitates three times with IP buffer, SDS-PAGE and western blot were performed for analysis of the immunoprecipitated proteins, as described. For Western blot analysis, embryos were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 10 l/ml protease inhibitor cocktail (Merck Millipore) and cellular debris cleared by centrifugation. Protein concentration in the lysate was determined using the BCATM protein assay kit (Thermo Scientific) according to the manufacturer’s instructions. Precipitated protein were then separated by SDS-PAGE on 10% gel. Proteins were transferred to PVDF membranes (Roche). After blocking in 5% BSA for 3 hour, proteins were detected by incubation with primary antibodies overnight at 4 °C followed by HRPconjugated secondary antibodies before detection by Image Quant LAS-4000 mini (GE Healthcare). Primary antibody were used srGAP2 (abcam, Cambridge, UK), Sox-19a (AnaSpec), alpha catenin (Acris Antibodies GmbH). 3 Legend of Supporting Information Tables Supporting Information Table 1. List of all identified phosphoproteins in (A) 12, (B) 24, (C) 48, and (D) 72 hpf embryos mixed with 120 hpf embryos, showing peptide sequence, UniProtKB ID, ratio of light and heavy, ion score, charge. (E) List of all quantified and non-quantified phosphopeptides with UniProtKB ID, protein quantitation ratio, GO and KEGG pathway enrichment. Supporting Information Table 2. Distribution of phospho-Ser/Thr/Tyr residues in different species Supporting Information Table 3. Distribution of phosphopeptides and motifs in zebrafish embryos. (A) Phosphorylated site motif; (B) motif classes Supporting Information Table 4. List of related CK2 proteins identified during embryonic development in zebrafish 4 Supporting Information Table 2. Distribution of phospho-Ser/Thr/Tyr residues in different species. Organism pSer % pThr % pTyr % Reference H. sapiens 86.4 11.8 1.8 (22) M. musculus 87.8 10.3 1.9 (23) D. melanogaster 78.7 18.5 2.8 (24) A. thaliana 84.7 13.6 1.7 (14) 5 Supporting Information Table 4. List of related CK2 proteins identified during embryonic development in zebrafish. Protein Quantitation Ratio UniProt # Phospho Position Motif Logo Protein description Dynamic Cluster 12 h 24 h 48 h 72 h A1L220 17 ******SDEE*** Smc4 protein (Fragment) Cluster1 3.892 4.594 4.496 3.209 A4FUK3 72 ******SDDD*** Si:dkeyp-55f12.3 Cluster1 2.342 2.6 - 1.765 F1QVR7 581 ******SDEE*** Protein SDA1 homolog Cluster1 7.458 10.036 - 5.237 F8W2Y9 643 ******SDDE*** Ubiquitin carboxyl-terminal hydrolase Cluster1 2.035 2.151 3.017 1.558 34 ******SEEE*** Cluster1 172 ******SEEE*** Cluster1 200 ******SEEE*** 2.907 2.869 3.678 2.513 212 ******SDEE*** 237 ******SDDD*** Cluster1 1.671 1.621 1.659 1.239 Cluster1 2.614 3.014 4.117 2.351 Q08CQ9 Zgc:152810 Cluster1 Cluster1 Cluster1 Q5SP50 153 ******SEEE*** Q7ZUT3 18 ******SDEE*** Zinc finger CCHC-type and RNA-binding motifcontaining protein 1 Dkc1 protein (Fragment) A1L227 25 ******SDDE*** Zgc:158606 Cluster2 4.175 2.9 3.64 3.061 A3KNV7 59 ******SDEE*** Si:dkeyp-35b8.5 protein (Fragment) Cluster2 11.634 10.075 8.605 4.801 90 ******SEEE*** 901 ******SEDE*** 13.821 4.747 3.85 2.418 Q6DHE3 308 ******SDDE*** Zgc:136471 protein (Fragment) Cluster2 11.743 9.187 6.853 7.644 Q7SXG4 599 ******SDEE*** SUMO-activating enzyme subunit 2 Cluster2 4.459 - 3.388 2.387 Q7ZTI0 364 ******SDEE*** Sjogren syndrome antigen B (Autoantigen La) Cluster2 6.496 4.79 4.941 2.848 Q7ZV16 107 ******SDEE*** Eukaryotic translation elongation factor 1 beta 2 Cluster2 2.152 1.782 1.342 1.388 Q7ZWA9 385 ******SEEE*** Eukaryotic translation initiation factor 5 Cluster2 2.932 2.357 2.045 1.964 Q8JIU7 166 ******SEEE*** Nascent polypeptide-associated complex subunit alpha Cluster2 15.938 2.114 4.653 1.774 Q1L8U8 Histone-lysine N-methyltransferase SETDB1-A 6 Cluster2 Cluster2 Supporting Information Figures - Deyolking + Protein 170kDa 130kDa 95kDa 72kDa 55kDa 43kDa 34kDa 26kDa 1 2 3 4 5 6 Supporting Information Figure S1. To confirm removal of yolk proteins, increasing amounts of 12 hpf embryo protein samples from zebrafish embryos were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before (lanes 1 to 3) and after deyolking (lanes 4 to 6). 7 258,180 MS/MS spectrum (search input) Mascot 2.3 with Proteome discoverer 1.3 9,564 unique phosphopeptides 4,866 Non-redundant phosphopeptides (Mascot score ≥ 20, False discovery rate ≤ 1%) Phosphorylation bioinformatics analysis 2,166 Phosphoproteins 1,564 Quantified phosphoproteins PhosphoRS (Site localozation) 3,500 Non-redundant high confidently phosphorylation sites (3,113 Serine, 356 Therosin, 31 Tyrosine) (pRS probability ≥ 75%) Motif-X analysis Supporting Information Figure S2. Schematic illustration of large-scale phosphorylation site identification from zebrafish embryos. A total of 258,180 MS/MS spectra were searched against zebrafish proteins using Mascot with Proteome Discoverer. A total of 9,564 unique phosphopeptides and 4866 non-redundant phosphorylation sites were identified with a Mascot score of more than 20, FDR less than 1%. A total of 3,500 high-confidence phosphorylation sites were filtered from the 4,866 non-redundant phosphorylation sites using a phosphoRS probability of more than 75%. Finally, phosphorylation motifs were extracted from these 3,500 high-confidence phosphorylation sites. 8 (B) (A) Serine, 88.9% Single, 91.4% Triply or more, 0.7% Tyrosine, 0.9% Threonine, 10.2% Doubly, 7.9% (D) (C) Proline-directed, 45.9% No Motif, 19.8% Others, 10.1% Motif, 80.2% Basophilic, 17.2% Acidophilic, 26.8% Supporting Information Figure S3. Distribution of phosphopeptide and motif in zebrafish embryos. (A) Diagram of the mono- or multi- phosphorylation of unique phosphopeptides. (B) Diagram of Ser/Thr/Tyr nonredundant phosphorylation sites in non-redundant phosphopeptides. (C) Classification of phosphorylation sites into motif classes. (D) Classification of the phosphorylation sites into 4 predominant motif classes. 9 Developmental stage Supporting Information Figure S4. Distribution of enriched motif during development stages. 10 (B) All-Ser/Thr/Tyr Phospho-Ser/Thr/Tyr 100 80.7 83.2 80 60 40 20 0 13.1 10.4 -Helix 6.2 6.4 -Strand Coil Mean gene homology percentage (%) Mean secondary structure percentage (%) (A) 100 100 100 All proteins Phosphoproteins 80 69.1 69.1 60 55.6 49.6 49.6 45.9 40 35.6 28.7 20 25.4 15.6 0 Zebrafish H. sapiens M. musculus D.melanogaster C.elegans S.cerevisiae (C) Supporting Information Figure S5. Characterization of phosphopeptides in zebrafish embryos. (A) Distributions of phospho-Ser/Thr/Tyr and non phospho-Ser/Thr/Tyr amino acids in protein secondary structures. (B) Proportions of zebrafish proteins exhibiting homology with H. sapiens, M. musculus, D. melanogaster, C. elegans, and S. cerevisiae. (C) GO analysis classification of phosphoproteins and proteins in zebrafish 11 (A) (B) (C) Supporting Information Figure S6. Confirming dynamic phosphoproteins during zebrafish development using GO annotation and a heat map. (A) Molecular function; (B) biological process; (C) cellular component. 12 12 hr / 120 hr ◆ 100 50 ■ 0 24 hr / 120 hr 100 ◆ Relative Abundance 50 ■ 0 100 48 hr / 120 hr 50 ◆ ■ 0 72 hr / 120 hr 100 ◆ ■ 50 0 735 736 737 738 739 m/z Supporting Information Figure S7. An example of a phosphopeptide that exhibited changes in phosphorylation level during embryonic development. EATPpTADSNSTAPK from the brain acid soluble protein 1 homolog (UniProt # Q1RM09) showed down-regulation during development. (■: 735.31 m/z, 737.32 m/z) 13 ◆: 120 A A Relative CK2 activity(%) 100 80 B 60 C D 40 20 0 12 hf 24 hf 48 hf 72 hf 120 hf Supporting Information Figure S8. CK2 activity during zebrafish development. The mean value ± standard error (S.E.) was determined for each treatment group of a given experiment. Difference between the means of the individual groups were assessed by oneway analysis of variance (ANOVA) with Duncan’s multiple ranges tests (SPSS) and differences of P<0.05 were considered significant. 14 IP : phospho-serine IB: α-catenin IB: SOX-19α IB: srGAP2 24 48 72 120 Time (hpf) Supporting Information Figure S9. Illustration of phosphoproteins in zebrafish embryos. The protein extracts (500 g of total protein) were immunoprecipited by anti-phosphoserine antibody and precipitated protein separated by SDS-PAGE on 10% gel. Samples were immunoblotted with the anti-srGAP2 (140 kDa, abcam), anti-Sox-19a (35 kDa, AnaSpec), anti-alpha catenin (102 kDa, Acris ntibodies GmbH). 15