Derived Peptide Libraries

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Determination of CK2 Specificity and Substrates by Proteome-derived Peptide Libraries Chunli Wang, Mingliang Ye, Yangyang Bian, Fangjie Liu, Kai Cheng, Mingming Dong, Jing Dong, and Hanfa Zou J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr4002965 • Publication Date (Web): 28 Jun 2013 Downloaded from http://pubs.acs.org on July 4, 2013

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Journal of Proteome Research

Determination of CK2 Specificity and Substrates by Proteome-derived Peptide Libraries Chunli Wang1, 2, Mingliang Ye1*, Yangyang Bian1, 2, Fangjie Liu1, 2, Kai Cheng1, 2, Mingming Dong1, 2, Jing Dong1, Hanfa Zou1 1 Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; 2 Graduate School of Chinese Academy of Science, Beijing 100049, China. KEYWORDS Peptide Library• Casein Kinase 2• Specificity• Substrates• Quantitative Phosphoproteomics

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ABSTRACT Understanding the specificity of kinases enables prediction of their substrates and uncovering kinase functions in signaling pathways. Traditionally synthesized peptide libraries are used to determine the kinase specificity. In this study, a proteomics-based method was developed to determine the specificity of kinase by taking the advantages of proteome-derived peptide libraries and quantitative proteomics. Proteome-derived peptide libraries were constructed by digesting proteins in total cell lysate followed with dephosphorylation of the resulted peptides. After incubating the peptide libraries with/without CK2 for in vitro kinase assay, stable isotopic labeling based quantitative phosphoproteomics was applied to distinguish the in vitro phosphosites generated by CK2. By using the above approach, 404 CK2 in vitro phosphosites were identified by 1D LC-MS/MS. Those sites allowed the statistic determination of the CK2 specificity. In addition to the easy construction of the proteome-derived peptide library, another significant advantage of this method over the method with synthesized peptide libraries is that the identified phosphosites could be directly mapped to proteins for the screening of putative kinase substrates. It was found that the confidence for substrate identification could be significantly improved by comparing the in vitro CK2 sites with the in vivo sites identified by phosphoproteomics analysis of the same cell lines. By applying this integrated strategy, 138 phosphosites from 105 putative CK2 substrates of high confidence were determined.


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INTRODUCTION Protein phosphorylation as an important post-translational modification is involved in almost all of cellular processes, including cell growth, differentiation, metabolism, proliferation and death1. The human genome encodes 518 human kinases, and only a few kinases have been characterized carefully and thoroughly2. Usually, different kinases targeted different sets of substrates. And this exquisite specificity between kinases and substrates is based on the structure of the catalytic site, local and distal interactions between the kinase and substrate, and other scaffolding and adaptor proteins3. The most important character of this recognition locates in the consensus sequences called motifs, such as K/R-X1-2-S/T for cAMP-dependent protein kinase (PKA)4. Understanding the specificity of kinases enables prediction of their substrates and uncovering kinase functions in signaling pathways. Protein kinase CK2 is an acidic-directed kinase that prefers acidic residues like Asp (D) or Glu (E) residues close to the modified residue. CK2 are overexpressed and hyperactivated in malignant tumors and cancers, which could be used for drug targets and prognosis of diseases5. It is reported that inhibition of CK2 kinase induces apoptosis of chronic lymphocytic leukemia cell, which is mediated by inactivation of PKC, a downstream PI3K target6. Besides, as the most prominent kinase with overlapping consensus sequence with caspase recognition motifs, CK2 regulates apoptotic progression by crosstalking with caspase7. Therefore studying specificity and substrates of CK2 has profound effects on CK2-dependent phosphorylation signaling pathways.

Many attempts have been made to dig out the specificity of kinase at peptide level8-10. Synthesized peptide library is often used to determine the kinase motifs. Although high-effective and high-throughput, it is labor-intensive and time-consuming to synthesize peptides. Another



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disadvantage is that synthesized peptide libraries lack database-searchable sequences and could not be efficiently identified by LC-MS/MS through proteomics platform11. However, proteomederived peptide libraries contain enormous array of natural and biological sequences so that it is amenable to mass spectrometry identification and database-search platform. Besides, it is easy to construct proteome-derived peptide library from any species of interested. Proteome derived peptide libraries have been applied to determine the cleavage specificity of proteases12. Our preliminary study indicated that the proteome derived peptide libraries can also be used to determine kinase motifs13. However, the endogenous sites interferes the determination of the sites phosphorylated by kinase. Therefore, an effective method to differentiate the kinase in vitro phosphosites with the background phosphosites is required.

In this study, stable isotopic labeling based quantitative proteomics coupled with proteome derived peptide libraries was applied to determine the specificity of kinase. The application of quantitative proteomics allowed accurately determining phosphosites generated by the kinase of interest. This approach was applied to determine CK2 specificity where 404 CK2 in vitro phosphosites were successfully identified. Inspired by the method developed by Tao et al14 that using kinase assay at peptide level linked with phosphoproteomics for identifying direct substrates of protein kinases, we further extended this peptide libraries method to screen CK2 substrates. The identified in vitro CK2 phosphosites were directly mapped to proteins for the screening of putative kinase substrates. It was found that the reliability of substrate identification could be improved significantly by filtering with in vivo phosphoproteome dataset.

EXPERIMENTAL DETAILS

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Human Jurkat T cell line (TCHU123, ATCC) was purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). RPMI 1640 medium, penicillin and streptomycin were obtained from Gibco Invitrogen Corporation (Carlsbad, CA). Fetal bovine serum was obtained from Biochrom AG (Berlin, Germany). Sodium bicarbonate (NaHCO3), sodium chloride (NaCl), HEPES, ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), iodoacetamide (IAA), trifluoroacetic acid (TFA), formic acid (FA), protease inhibitor cocktail, alkaline phosphatase (AP), sodium cyanoborohydride (NaBH3CN), formaldehyde solution (CH2O, 37%) and Formaldehyde-d2 solution (CD2O, 20%) were obtained from Sigma (St. Louis, MO). Phenylmethylsulfonyl ﬂuoride (PMSF) were purchased from AMERESCO (Solon, Ohio, USA). Sequencing trypsin was obtained from Promega (Madison, WI, USA). Phosphatase inhibitor cocktail tablets (PhoSTOP) were purchased from Roche (Mannheim, Germany). Acetonitrile (ACN) and 25% ammonia solution (NH3•H2O) were purchased from Merck (Darmstadt, Germany). Casein kinase 2 (CK2) was obtained from Millipore (Bedford, MA, USA). HLB C18 cartridges were provided by Waters (Milford, MA). All the water used in the experiments was prepared using a Milli-Q system (Millipore, Bedford, MA). All the chemicals were of analytical grade except acetonitrile, which was of HPLC grade.

Cell culture and protein extraction

Jurkat cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum at 37 ℃ under 5% CO2, 100 unit/mL penicillin and 100 µg/mL streptomycin. Cells were collected at a density of 106 cells/mL by centrifugation at 1000 g and washed with ice-cold PBS for three times. Then the cells were resuspended in an ice-cold lysis buffer containing 8 M Urea in 50 mM Tris-HCl (pH=7.4), 65 mM DTT, 1 mM EDTA, 1% (v/v) Triton, 1 mM PMSF (added freshly), 2%



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(v/v) protease inhibitor cocktail (added freshly) and phosphatase inhibitor cocktail (1 tablet/10 mL lysis buffer, added freshly), and ultrasonicated in an ice-bath. The lysate was centrifuged at 20000 g for 20 min at 4 ℃, and the supernatant was collected and to be precipitated with 5 volumes of ice-cold solution containing 50% acetone, 50% alcohol and 0.1% acetic acid at -20 ℃ overnight, and lyophilized to dryness. The proteins were re-dissolved in a buffer containing 8 M Urea, 50 mM Tris-HCl (pH= 8.3) and the concentration of proteins was 8.3 mg/mL measured by BCA method. And the re-dissolved proteins solution was stored at -80 ℃ for further analysis.

2 mg above re-dissolved proteins were reduced by 20 mM DTT at 37 ℃ for 2 h and oxidized by 40 mM IAA at 25 ℃ for 45 min in dark. The protein mixture was further diluted to 2 mL with 50 mM Tris-HCl buffer (pH=8.3). After adding 40 µg sequencing trypsin, the protein mixture was digested at 37 ℃ overnight. The obtained digests were kept at -80 ℃.

In vitro CK2 assay

Tryptic peptides digest (0.5 mg) was desalted by 10 mg-HLB C18 cartridges and was redissolved in 0.5 mL dephosphorylation reaction buffer containing 50 mM HEPES, 1 mM ZnCl2 and 1 mM MgCl2. After adding 50 U alkaline phosphatase (AP), the dephosphorylation reaction was proceeded at 37 ℃ overnight. After that, the solution was heated at 95 ℃ for 15 min to inactivate the activity of added alkaline phosphatase. Then the peptides solution was divided into two equal aliquots and separately diluted to 350 µL containing a final concentration of 0.1 mM EDTA, 100 mM NaCl, 5 mM DTT, 10 mM MgCl2 and 0.1% Triton in 50 mM HEPES ( pH=7.4). One was used for control where 100 µM ATP was added. The other was used for CK2 reaction


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where 100 µM ATP and 0.5 µg CK2 were added. The CK2 kinase catalyzed reaction was performed at 30 ℃ for 30 min.

Dimethyl labeling reaction and phosphopeptides enrichment

After kinase reaction, the control peptides and CK2 reaction peptides were separately labeled by an in-solution stable isotope dimethyl labeling method15. First, the control peptides and CK2 reaction peptides solution was separately diluted to 1 mL with 100 mM TEAB buffer (pH=8.0). Then 40 µL 4% CH2O and 40 µL 0.6 M NaBH3CN were added into the control peptides for light-labeling reaction while 40 µL 4% CD2O and 40 µL 0.6 M NaBH3CN were added into the CK2 reaction peptides for heavy-labeling reaction at 20 ℃ for 1 h. After that, 16 µL 10% NH3•H2O were added to terminate the labeling reaction followed with addition of 20 µL TFA to neutralize the solution. Finally, the two samples were mixed together for phosphopeptides enrichment following the protocol described by Yu et al16. Briefly, the combined labeled peptides were mixed with equal volume of 5 mg Ti4+-IMAC microsphere suspension solution (6% TFA, 50% ACN) and vibrated for 30 min. Then the microspheres were washed with solution containing 50% ACN, 6% TFA, 200 mM NaCl and followed with washing solution containing 30% ACN, 0.1% TFA with vibration for 15 min to remove unphosphorylated peptides. The bound phosphopeptides were eluted with 500 µL of 10% NH3•H2O with vibration and sonication for 15 min respectively. The eluted phoshopeptides were lyophilized to dryness and resuspended in 100 µL of 0.1% FA for further MS analysis.

Preparation of samples for proteome and phosphoproteome analysis of Jurkat T cell



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For proteome analysis, tryptic peptide from the digest of cell lystate of Jurkat cells (0.5 mg) was desalted by 10 mg-HLB C18 cartridges and was re-dissolved in 250 µL of 0.1% FA. For phosphoproteome analysis, tryptic peptides from the digest of cell lystate of Jurkat cells (1 mg) was acidified by 10% TFA and then the phosphopeptides were directly enriched by 10 mg Ti4+IMAC microspheres following the above enrichment procedure. And the resulted phosphopeptides were re-suspended in 100 µL of 0.1% FA.

Mass Spectrometry Analysis

Enriched phosphopeptides were analyzed on LTQ-Orbitrap-Velos mass spectrometer (Thermo, San Jose, CA) with 1D RP-LC separating system in the positive ion mode. Phosphopeptides (50 µg) sample in 0.1% FA were loaded onto C18 trap column, and then separated by a homemade C18 capillary column (75 µm id × 150 mm length) with a 135 min RP gradient elution. A Finnigan surveyor MS pump (Thermo, San Jose, CA) was used to deliver the mobile phase consisted of mobile A, 0.1 % (v/v) formic acid in water, and mobile phase B, 0.1% (v/v) formic acid in ACN. The flow rate was adjusted to 200 nL/min after splitting. The gradients elution was performed with gradients of 0-2% B in 2 min, 2-25% B in 93 min, 25-35% B in 10 min, 35-80% B in 2 min, 80% B in 10 min, 80% B-100% A in 3 min and 100% A in15 min.

Multistage activation (MSA) method was used for phosphopeptides analysis17. The temperature of the ion transfer capillary was 250 ℃, the electrospray voltage was +2.0 kV, the normalized collision energy was 35%, and the activation time was 10 ms. Mass spectrometry was operated in data-dependent MS/MS acquisition mode. Full mass scan performed in the Orbitrap analyzer was acquired from m/z 400 to 2000 (R = 60000 at m/z 400) and MS/MS scan was acquired in LTQ linear ion trap. One microscan was set for each MS and MS/MS scan. All MS and MS/MS 8 Environment ACS Paragon Plus

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spectra were acquired in the data-dependent analysis mode, in which the 20 most intense ions with MS scan were selected for MS/MS scan by collision-induced dissociation (CID) with multistage activation on. The neutral loss mass list was set as follows: 98, 58, 49, 38.67, 32.67 and 24.5. The dynamic exclusion function was set as follows: repeat count 2, repeat duration 30 s, and exclusion duration 60 s.

For Jurkat proteome analysis, we used LTQ-Orbitrap-Velos mass spectrometer with 2D SCXRP-LC separating system. 20 µg tryptic peptides were loaded onto SCX monolithic trap, and then a series of salt steps elution were applied to fractionate the peptides followed with an RP gradient separation as above. The NH4Ac (pH 2.7) salt steps were set as follows: 50, 100, 150, 200, 250, 300, 350, 400, 500 and 1000 mM. Each salt step lasted 10 min with additional 15 min 0.1% FA for equilibration. The mass spectrometry parameters were set as the above except multistage activation turned off and neutral loss parameters discarded.

Two technical replicates of each sample were carried out.

Database searching and quantification

All MS/MS spectra were searched using Maxquant (version1.1.1.36) against the international protein index (IPI) database (IPI human 3.80) with 1% false discovery rate (FDR). The parameters were set as follows: precursor-ion mass tolerance, 10 ppm; fragment-ion mass tolerance, 0.8 Da; enzyme, trypsin (KR/P); missed cleavage, 2; static modification, Cys (+57.02146 Da); dynamic modifications methionine (+15.99491 Da). All the technical replicates of the same sample were integrated into one experiment for database searching.



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For the searching the spectra of phosphopeptides, Ser, Thr and Tyr (+79.96633 Da) were set as dynamic modifications. For the quantification analysis, double labeling parameters were added as follows: lysine and peptide amino termini in light dimethylation (+28.03130 Da) and heavy dimethylation (+32.05641 Da).

RESULTS AND DISCUSSIONS

Distinguishing of in-vitro CK2 phosphosites by quantitative proteomics

The workflow for determination of CK2 specificity using proteome-derived peptide libraries is given in Fig.1. In general, it has three steps, i.e. peptide libraries construction, in-vitro kinase reaction and phosphosite quantification. At first, the proteins in cell lysate are digested with trypsin. The generated peptides contain many endogenous phosphopeptides. The peptides from bona fide CK2 substrates are likely already phosphorylated in vivo and cannot be phosphorylated by in vitro reaction any more. To use the tryptic peptides as the peptide library for the determination of kinase specificity, the resulted tryptic peptides must be dephosphorylated by alkaline phosphatase. After dephosphorylation, the peptides are divided into two equal aliquots. One aliquot is incubated with CK2 and ATP for kinase reaction, and the other aliquot is incubated with ATP as the control experiment to determine the background phosphosites. After reactions, above two aliquots are separately labeled with heavy and light stable isotopic dimethyl labeling reagents. Then, the labeled peptides are combined together and subjected to IMAC enrichment. The enriched phosphopeptides are analyzed by LC-MS/MS following with quantitative proteomics approach. The in-vitro sites phosphorylated by CK2 are finally determined by phosphopeptide ratios between the kinase reaction group and the control group.


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In this study, the tryptic peptides derived from Jurkat cells were used as the peptide libraries for the determination of CK2 specificity. Since alkaline phosphatase could not completely remove the endogenous kinase interference13, we introduced the dimethyl labeling method to differentiate the remaining endogenous phosphopeptides from the phosphopeptides generated by the in vitro reaction. After analysis following above workflow, 558 unique phosphosites from 353 proteins were quantified in 2 replicated MS runs (Supplementary Table S1). In quantitative proteomics, ratios over two folds are generally considered as significantly changed. According to the log2Ratio distribution of quantified phosphosites in Fig.2A, we classified the quantification phosphosites into three groups: significantly up-regulated sites (log2R≥1), no significant changed sites (-1

Derived Peptide Libraries

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