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.
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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.
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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).
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