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Chemical Genetic Approach for Kinase‐Substrate Mapping by Covalent Capture of Thiophosphopeptides and Analysis by Mass Spectrometry

Nicholas T. Hertz1,2,  Beatrice T. Wang2,  Jasmina J. Allen1,2,  Chao Zhang2,3,  Arvin C. Dar2,  Alma L. Burlingame4,  Kevan M. Shokat2,3

1Chemistry and Chemical Biology Graduate Program, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California
2Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California
3Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California
4Department of Pharmaceutical Chemistry, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California




Publication Name: 
Current Protocols in Chemical Biology
Unit Number: 
 
DOI: 
10.1002/9780470559277.ch090201
Online Posting Date: 
February, 2010
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Abstract

Mapping kinase-substrate interactions demands robust methods to rapidly and unequivocally identify substrates from complex protein mixtures. Toward this goal, we present a method in which a kinase, engineered to utilize synthetic ATPS analogs, specifically thiophosphorylates its substrates in a complex lysate. The thiophosphate label provides a bio-orthogonal tag that can be used to affinity purify and identify labeled proteins. Following the labeling reaction, proteins are digested with trypsin; thiol-containing peptides are then covalently captured and non-thiol-containing peptides are washed from the resin. Oxidation-promoted hydrolysis, at sites of thiophosphorylation, releases phosphopeptides for analysis by tandem mass spectrometry. By incorporating two specificity gates—kinase engineering and peptide affinity purification—this method yields high-confidence substrate identifications. This method gives both the identity of the substrates and phosphorylation-site localization. With this information, investigators can analyze the biological significance of the phosphorylation mark immediately following confirmation of the kinase-substrate relationship. Here, we provide an optimized version of this technique to further enable widespread utilization of this technology. Curr. Protoc. Chem Biol. 2:15-36. © 2010 by John Wiley & Sons, Inc.

Keywords: phosphorylation; chemical genetics; analog specific kinase; kinase substrate identification; thiophosphate labeling

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol: Digestion and Covalent Capture of Thiophosphorylated Peptides
  • Support Protocol 1: Kinase Reaction with ATPS Followed by Immunoblotting Utilizing Thiophosphate-Specific Antibody
  • Alternate Protocol 1: Identifying Optimal N6-Substituted ATPS Analog
  • Support Protocol 2: Thiophosphorylation of a Candidate Kinase Substrate in Cell Lysate
  • Support Protocol 3: Preparation of a Thiophosphorylated Positive Control Peptide and Protein
  • Support Protocol 4: Chemical Synthesis of N6-Substituted ATPS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Digestion and Covalent Capture of Thiophosphorylated Peptides

 Materials
  • 2× denaturation buffer (see recipe)
  • 1 M tris(2-carboxyethyl)phosphine (TCEP) in H2O; store up to 6 month at –80°C
  • Urea (99% pure); store indefinitely at room temperature
  • Lysate to be analyzed (Strategic Planning)
  • ATPS analog (Support Protocol 4; several N6-substituted ATPS analogs are also available from Biolog, http://www.biolog.de)
  • AS kinase of interest (KOI: typically prepared in 100 mM Tris×Cl, pH 7.5/150 mM NaCl/1 mM DTT, which may be augmented with other reagents, depending on the kinase; see published literature)
  • Controls (these controls will ensure that all the steps of this protocol are working correctly; the MBP alone and MBP plus lysate will lead to recovery of only one peptide (see Anticipated Results); the lysate controls will provide a list of nonspecific substrates):
    • 100 pmol of hyper-thiophosphorylated MBP (see Support Protocol 3)
    • Unlabeled lysate: same lysate used for labeling, with no kinase or ATP added (a normal starting point is to use 1 mg total protein lysate in 100 µl total volume of 100 mM Tris×Cl, pH 7.5/150 mM NaCl/1 mM DTT/10 mM MgCl2 with protease inhibitors)
    • Unlabeled lysate plus 100 pmol hyper-thiophosphorylated MBP
    • Lysate plus ATPS analog minus kinase
    • An additional covalent capture reaction control (see step 7) with CREB peptide (see Support Protocol 3)
  • Experimental sample: lysate plus ATPS analog plus AS kinase (the amount of AS kinase should be ~1% with respect to the mass of protein in the lysate, e.g., 10 µg AS kinase/1 mg protein; using the thiophosphate ester–specific western blot, looking for which conditions give the least nonspecific background and best labeling can optimize the ratio; Shah et al., 1997)
  • 50 mM NH4HCO3
  • Trypsin (Promega, cat. no. V5113)
  • 2.5% (v/v) trifluoroacetic acid (TFA)
  • 0.1% TFA/50% acetonitrile in H2O (store indefinitely at room temperature)
  • 0.1% TFA in H2O (store indefinitely at room temperature)
  • Iodoacetyl agarose beads, 50% slurry (Pierce; store up to 6 months at 4°C)
  • 200 mM HEPES, pH 7.0 (store up to 4 months at 22°C)
  • 50% (v/v) acetonitrile/50% (v/v) 20 mM HEPES, pH 7.0 (store up to 4 months at 22°C)
  • 5 mg/ml bovine serum albumin (BSA)
  • Acetonitrile
  • 50% (v/v) acetonitrile/50% (v/v) H2O (store indefinitely at room temperature)
  • 5 M NaCl (store indefinitely at room temperature)
  • 5% (v/v) formic acid (store up to 3 months at room temperature)
  • 1 M dithiothreitol (DTT; store up to 6 months at –80°C)
  • Oxone (DuPont)
  • Siliconized microcentrifuge tubes
  • 55°C water bath
  • C-18 Sep Pak column (Sep Pak Classic cartridge; total volume, 0.5 ml; Waters; store indefinitely at room temperature in desiccator); alternatively use Oasis SPE (Waters)
  • Small disposable columns (Isolute SPE Accessories Double Fritted Column 120-1021-A and Single Fritted Res 120-1111-A; Biotage, http://www.biotage.com/)
  • 10- and 100-µl C-18 ZipTips (Millipore)
  • QSTAR Elite Mass spectrometer (Applied Biosystems) or other tandem LC MS/MS capable mass spectrometer (also see Carr and Annan, 1996)
  • Mass spectrometry analysis software (Carr and Annan, 1996)
  • Additional reagents and equipment for mass spectrometry (Carr and Annan, 1996)

Support Protocol 1: Kinase Reaction with ATPS Followed by Immunoblotting Utilizing Thiophosphate-Specific Antibody

 Materials
  • 1× HEPES-buffered saline (HBS; see recipe for 10×) or other kinase reaction buffer suitable for the KOI
  • 1 M MgCl2
  • Kinase substrate (or general kinase substrate, e.g., myelin basic protein or histone H1)
  • Kinase of interest (KOI)
  • N6-substituted adenosine 5¢-[-thio]triphosphate (exclusively from Biolog: http://www.biolog.de), store 10 mM stock in aliquots at –80°C for up to 1 year and avoid freeze-thaw cycles
  • p-nitrobenzyl mesylate (PNBM; exclusively from Epitomics, http://www.epitomics.com); store solid for up to 1 year at 4°C (50 mM stock in DMSO should be prepared fresh just before use)
  • 5× sample buffer (Gallagher, 2006)
  • 5% skim milk in TBST (see recipe for TBST)
  • Primary antibody: thiophosphate ester rabbit monoclonal antibody, clone 51-8 (exclusively from Epitomics, http://www.epitomics.com; store up to 1 month at 4°C or indefinitely at –20°C)
  • Secondary antibody: anti-rabbit HRP-conjugated antibody (Epitomics, http://www.epitomics.com); store up to 1 month at 4°C or indefinitely at –20°C
  • ECL detection system
  • Additional reagents and equipment for SDS-PAGE (Gallagher, 2006) and immunoblotting (Gallagher et al., 2008)

Support Protocol 2: Thiophosphorylation of a Candidate Kinase Substrate in Cell Lysate

 Materials
  • Appropriate mammalian cells
  • Plasmid containing (wild-type or AS) KOI suitable for expression of kinase in mammalian cells
  • Transfection reagents (e.g., Lipofectamine, FuGene; see manufacturer's protocol for transfection of plasmid)
  • Phosphate buffered saline (PBS), store indefinitely at 4°C
  • 2× RIPA buffer (see recipe)
  • Protease inhibitor cocktail without EDTA (Roche); store up to 1 year at 4°C
  • Phosphatase inhibitor cocktail (Roche); store up to 1 year at 4°C
  • ATP (Sigma; store solid indefinitely at –20°C)
  • GTP (Sigma; store solid indefinitely at –20°C)
  • N6-substituted adenosine 5¢-[-thio]triphosphate (exclusively from Biolog: http://www.biolog.de), store 10 mM stock in aliquots at –80°C for up to 1 year and avoid freeze-thaw cycles
  • Disodium EDTA (Sigma), store solid indefinitely at room temperature
  • p-nitrobenzyl mesylate (PNBM; exclusively from Epitomics, http://www.epitomics.com); store solid for up to 1 year at 4°C (50 mM stock in DMSO should be prepared fresh just before use)
  • Protein A or G Magnetic beads (Invitrogen, Dynabeads); store up to 1 year at 4°C
  • 5× sample buffer (Gallagher, 2006)
  • 10-cm cell culture dishes
  • Cell scrapers
  • End-over-end rotator
  • Magnetic stand that holds 1.5-ml microcentrifuge tubes
  • Additional reagents and equipment for SDS-PAGE and immunoblotting (Support Protocol 1)

Support Protocol 3: Preparation of a Thiophosphorylated Positive Control Peptide and Protein

 Materials
  • 10× HEPES-buffered saline (HBS; see recipe)
  • 1 M MgCl2
  • 10 mM stock in H2O of adenosine 5¢-[-thio]triphosphate tetralithium salt (Sigma), store up to 1 year at –80°C
  • Substrates:
    • CREB peptide (KRREILSRRPS(p)YR; 2 nmol/µl), store up to 1 year at –80°C
    • Dephosphorylated myelin basic protein (2.5 mg/ml) (Millipore)
  • Purified active KOI or plasmid containing KOI suitable for expression of kinase in E.coli
  • Recombinant GSK3 (Millipore)
  • Liquid N2
  • 0.1% TFA/50% acetonitrile in H2O (store indefinitely at room temperature)
  • 0.1% TFA in H2O (store indefinitely at room temperature)
  • OMIX 100-µl ZipTips (Varian)

Support Protocol 4: Chemical Synthesis of N6-Substituted ATPS

 Materials
  • 6-chloropurine ribonucleoside (Sigma, cat. no. C8276-56)
  • Ethanol (Sigma)
  • Alkylamine containing desired N6 modification: e.g., phenethylamine
  • Triethylphosphate (TEP; Sigma)
  • POCl3 (Sigma)
  • H3PO4 (Sigma)
  • 1,8-diazabicyclo[5.4.0]undec-7-en (DBU) (Sigma)
  • 2 M TEAB (see recipe)
  • Trisodium thiophosphate (Sigma-Aldrich)
  • 3-chloropropionamide (Sigma-Aldrich)
  • Dimethylformamide (DMF)
  • DOWEX 501-X8 ion exchange resin (pyridinium form)
  • Pyridine, dry
  • Methanol
  • Tri-n-octylamine (Sigma-Aldrich)
  • Dioxane, dry
  • Diphenyl phosphorochloridate (Sigma-Aldrich)
  • Tri-n-butylamine (Sigma-Aldrich)
  • Diethyl ether
  • Petroleum ether
  • 0.2 M NaOH
  • -mercaptoethanol
  • Oil bath
  • Reflux condenser
  • Buchi Rotovapor Model R-200 or equivalent rotary evaporator
  • PTFE syringe filter (0.45 µm Pall Acrodisc)
  • HiPrep 16/10 QFF anion-exchange columns (Amersham Biosciences)
  • Peristaltic pump
  • ACTA FLPC system (GE Healthcare) including gradient former
  • Small molecule-capable LC-MS instrument (e.g., Waters; see Carr and Annan, 1996)
  • Lyophilizer
  • Filter paper
  • Buchner funnel
  • Small plastic column
  • Round-bottom flasks
  • High-vacuum source and high-vacuum manifold
  • Additional reagents and equipment for mass spectrometry (Carr and Annan, 1996)
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Figures

  • Figure 1.
    Thiophosphopeptide purification scheme. (A) Wild-type (wt) kinase utilizes ATP to phosphorylate its substrates. In contrast, an engineered analog-specific (AS) kinase has a new active-site pocket that allows it to accept an unnatural bulky ATP analog. This “lock and key” allows the AS kinase to transfer the thiophospho group to its substrates. (B) Detail of the two different affinity-purification methods. The thiophosphorylated substrates are found in a background of phosphorylated and unlabeled proteins. In the first technique, the thiophosphorylated proteins are reacted with a thiol-specific alkylating agent that generates a bio-orthogonal thiophosphate ester. Labeled proteins are detected by a thiophosphate ester-specific antibody. In the second approach, the lysate is first digested to generate tryptic peptides. Thiol-containing peptides are then captured by reaction with iodoacetyl agarose beads, and all non-thiol-containing peptides are washed away. The remaining peptides are treated with Oxone, which releases thiophosphate ester-linked peptides by spontaneous hydrolysis.

    View Image
  • Figure 2.
    (A) Labeling of a cell lysate by incubating AS kinase and N6-substituted ATPS analogs generates thiophosphorylated and phosphorylated proteins. (B) Alkylating thiol-containing proteins generates the bio-orthogonal thiophosphate ester. Thiophosphate ester–labeled proteins are detected by a thiophosphate ester–specific antibody (clone 51-8). (C) Thiol-containing peptides are captured by reaction with iodoacetyl beads. During washing, all other non-thiol-containing peptides are washed away. The bound peptides are treated with Oxone to convert the thiol group to a sulfoxide. The presence of an electrophilic phosphoester in the thiophosphate ester-linked peptides catalyzes the spontaneous hydrolysis of these peptides and, after concentration, these peptides are analyzed by tandem LC-MS/MS.

    View Image
  • Figure 3.
    (A) The gatekeeper residue is shown in CDK1 as a phenylalanine above the bound ATP in the CDK1 active site. After mutation to the much smaller glycine, a pocket is opened that allows for a bulky substituent to bind (pictured as asCDK1). A “bumped” (N6-substituted) ATP analog or inhibitor (in this case 1NMPP1) can now bind to the engineered kinase by utilizing the extra space above the N7 position. (B) The active sites of SRC, PKA, aurora, and ERK are shown along with their gatekeeper residues and the successful mutation of these bulky residues to smaller ones. The mutation of the gatekeeper to a smaller residue creates a similar pocket in each of these four kinases. These kinases are shown with ATP bound or with bumped inhibitors that target these engineered kinases, but not the wild type. The similarity in the engineered pocket among disparate kinases demonstrates the wide applicability of this method.

    View Image
  • Figure 4.
    Synthetic scheme for the production of N6-substituted ATPS analogs.

    View Image
  • Figure 5.
    (A) Raw data showing an expected MALDI analysis of thiophosphorylated CREB peptide. Both the singly phosphorylated (KRREILSRRPS(p)YR) (1795.84 MH+) and the thiophosphorylated (KRREILS(thiophosphate)RRPS(p)YR) 1891.986 (MH+) peptide are shown in the first pane. Analysis of the flow-through after covalent capture with iodoacetyl agarose beads in the second pane shows the complete reaction of the thiophosphorylated (1891.986) peptide, but no reaction with the parent peptide (1795.84 MH+). Analysis of the flow-through with additional HeLa lysate after covalent capture with iodoacetyl agarose beads in the third pane shows many peptides, but the CREB peptide is not seen. (B) Following hydrolysis, the only peptide present is the double phosphorylated CREB peak at 1875.986 (MH+), demonstrating the specificity of the technique. (C) After the covalent capture reaction with digested thiophosphorylated myelin basic protein, only one peptide (NIVT(phospho)PRTPPPSQGK) is observed. MS-MS analysis of the 1587.8001 (MH+), NIVT(phospho)PRTPPPSQGK (seen as the triply phosphorylated peptide at 524.6 m/z) shows good sequence coverage of this peptide with loss of phosphate from the y10 ion.

    View Image

Literature Cited

Literature Cited
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    Bishop, A., Buzko, O., Heyeck-Dumas, S., Jung, I., Kraybill, B., Liu, Y., Shah, K., Ulrich, S., Witucki, L., Yang, F., Zhang, C., and Shokat, K.M. 2000. Unnatural ligands for engineered proteins: New tools for chemical genetics. Annu. Rev. Biophys. Biomol. Struct. 29:577-606.
    Blethrow, J., Zhang, C., Shokat, K.M., and Weiss, E.L. 2004. Design and use of analog-sensitive protein kinases. Curr. Protoc. Mol. Biol. 66:18.11.1-18.11.19.
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