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

Nicholas T. Hertz1, Beatrice T. Wang1, Jasmina J. Allen1, Chao Zhang2, Arvin C. Dar1, Alma L. Burlingame3, Kevan M. Shokat2

1 Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, 2 Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, 3 Department 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 ATPγS 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 1: Digestion and Covalent Capture of Thiophosphorylated Peptides
  • Support Protocol 1: Kinase Reaction with ATPγS Followed by Immunoblotting Utilizing Thiophosphate‐Specific Antibody
  • Alternate Protocol 1: Identifying Optimal N6‐Substituted ATPγS 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 ATPγS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Digestion and Covalent Capture of Thiophosphorylated Peptides

  Materials
  • 2× denaturation buffer (see recipe)
  • 1 M tris(2‐carboxyethyl)phosphine (TCEP) in H 2O; store up to 6 month at −80°C
  • Urea (99% pure); store indefinitely at room temperature
  • Lysate to be analyzed (Strategic Planning)
  • ATPγS analog ( protocol 6; several N6‐substituted ATPγS 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 protocol 5)
    • 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 MgCl 2 with protease inhibitors)
    • Unlabeled lysate plus 100 pmol hyper‐thiophosphorylated MBP
    • Lysate plus ATPγS analog minus kinase
    • An additional covalent capture reaction control (see step 7) with CREB peptide (see protocol 5)
  • Experimental sample: lysate plus ATPγS 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., )
  • 50 mM NH 4HCO 3
  • Trypsin (Promega, cat. no. V5113)
  • 2.5% (v/v) trifluoroacetic acid (TFA)
  • 0.1% TFA/50% acetonitrile in H 2O (store indefinitely at room temperature)
  • 0.1% TFA in H 2O (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) H 2O (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, )
  • Mass spectrometry analysis software (Carr and Annan, )
  • Additional reagents and equipment for mass spectrometry (Carr and Annan, )

Support Protocol 1: Kinase Reaction with ATPγS 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 MgCl 2
  • Kinase substrate (or general kinase substrate, e.g., myelin basic protein or histone H1)
  • Kinase of interest (KOI)
  • N6‐substituted adenosine 5′‐[γ‐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, )
  • 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, ) and immunoblotting (Gallagher et al., )

Alternate Protocol 1: Identifying Optimal N6‐Substituted ATPγS Analog

  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′‐[γ‐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, )
  • 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 ( protocol 2)

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

  Materials
  • 10× HEPES‐buffered saline (HBS; see recipe)
  • 1 M MgCl 2
  • 10 mM stock in H 2O of adenosine 5′‐[γ‐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 GSK3β (Millipore)
  • Liquid N 2
  • 0.1% TFA/50% acetonitrile in H 2O (store indefinitely at room temperature)
  • 0.1% TFA in H 2O (store indefinitely at room temperature)
  • OMIX 100‐µl ZipTips (Varian)

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

  Materials
  • 6‐chloropurine ribonucleoside (Sigma, cat. no. C8276‐56)
  • Ethanol (Sigma)
  • Alkylamine containing desired N6 modification: e.g., phenethylamine
  • Triethylphosphate (TEP; Sigma)
  • POCl 3 (Sigma)
  • H 3PO 4 (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
  • β‐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, )
  • 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, )
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Figures

Videos

Literature Cited

Literature Cited
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