Mass Spectrometry‐Based Identification of Protein Kinase Substrates Utilizing Engineered Kinases and Thiophosphate Labeling

Yong Chi1, Bruce E. Clurman1

1 Divisions of Clinical Research and Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch100151
Online Posting Date:  November, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Protein kinases constitute a large enzyme family with key roles in cellular signal transduction. One way to elucidate the functions of protein kinases is to systematically identify their downstream targets. Presented here is a simple and effective method to identify direct protein kinase substrates in native cell lysates. First, the activity of the kinase of interest is isolated by engineering the normal kinase to utilize bulky ATP analogs that cannot be used by normal cellular kinases. This allows specific labeling of substrates with thiophosphate tags by performing kinase reactions in cell lysates that also include bulky ATP‐γ‐S analogs. After digesting the proteins in the reaction mixture, thiophosphopeptides are isolated using a single‐step capture‐and‐release protocol and identified by mass spectrometry. This technique is easy to use and generally applicable. Curr. Protoc. Chem. Biol. 2:219‐234 © 2010 by John Wiley & Sons, Inc.

Keywords: chemical genetics; analog‐sensitive kinase; thiophosphorylation; phosphopeptides

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Substrate Labeling and Purification of the Thiophosphopeptides
  • Support Protocol 1: Fractionation of HEK293 Cell Lysate
  • Support Protocol 2: Applying the Kinase Substrate Identification Method to a Control Substrate
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Substrate Labeling and Purification of the Thiophosphopeptides

  Materials
  • 5× kinase reaction buffer (see recipe)
  • Cell lysate (buffer composition similar to kinase assay condition; see protocol 2 and Strategic Planning)
  • Purified control substrate (protein known to be a substrate of the kinase of interest)
  • N6‐(2‐Phenylethyl)‐ATP‐γ‐S (TriLink BioTechnologies)
  • Kinase of interest
  • 0.5 M EDTA, pH 8 (MediaTech)
  • Acetonitrile (VWR)
  • Trypsin, sequencing grade (Promega)
  • 10% (v/v) formic acid (store in glass bottle at room temperature indefinitely)
  • ColorpHast pH‐indicator strips, pH 4.0 to 7.0 (Fisher Scientific)
  • Disulfide beads: thiopropyl‐Sepharose 6B (GE Healthcare)
  • Micro Bio‐Spin chromatography columns, 0.8 ml (Bio‐Rad Laboratories)
  • Washing solution 1 (see recipe)
  • Washing solution 2 (see recipe)
  • 20 mM sodium hydroxide (store at room temperature up to 3 months)
  • 1% (v/v) formic acid (store in glass bottle at room temperature indefinitely)
  • 0.6‐ and 1.7‐ml microcentrifuge tubes
  • Microcentrifuge, and mini‐microcentrifuge (e.g., VWR) for smaller tubes
  • 1‐ml syringes
  • 26‐G, 1/2‐in. needles
  • Labquake rotator (Fisher Scientific)
  • Iron stand and clamps (Fisher Scientific)
  • Dropper bulbs for 3‐ml Pasteur pipets (Fisher Scientific)
  • Tandem mass spectrometer (ThermoFisher Scientific)
  • Database search and data filtering software
  • Statistical software (see Deutsch, )

Support Protocol 1: Fractionation of HEK293 Cell Lysate

  Materials
  • HEK293 cells (ATCC, cat. no. CRL‐11268)
  • Phosphate‐buffered saline (PBS; MediaTech, cat. no. 21‐040‐CV)
  • Hypotonic lysis buffer (see recipe)
  • 5 M NaCl
  • BioRad protein assay dye reagent (BioRad Laboratories)
  • SP Sepharose Fast Flow resin (GE Healthcare)
  • Q Sepharose Fast Flow resin (GE Healthcare)
  • Column loading buffer (see recipe)
  • 30 mM Tris⋅Cl, pH 7.5
  • Column loading buffer (see recipe) containing 100 mM, 200 mM, 300 mM, 400 mM, and 600 mM NaCl
  • Ammonium sulfate (Fisher Scientific)
  • Dialysis buffer (see recipe)
  • 15‐cm cell culture dishes (Becton Dickinson)
  • 15‐ml and 50‐ml conical centrifuge tubes (e.g., BD Falcon, Fisher Scientific)
  • Tabletop centrifuge with swinging‐bucket rotor
  • Branson Sonifier 250 (Branson Ultrasonics)
  • 1.0 × 10 cm, glass chromatography columns (Bio‐Rad Laboratories)
  • Iron stands and clamps for chromatography columns
  • Accumet AB30 conductivity meter (Fisher Scientific)Refrigerated centrifuge
  • Amicon Ultra‐4 Centrifugal Filter, 10,000 MWCO (Millipore)
  • SnakeSkin pleated dialysis tubing, 7000 MWCO (Pierce)
  • Additional reagents and equipment for basic cell culture techniques including trypsinization (Phelan, )

Support Protocol 2: Applying the Kinase Substrate Identification Method to a Control Substrate

  Materials
  • GST‐Rb (expressed in E. coli and purified; Qin et al., ; contact Dr. Bruce Clurman, bclurman@fhcrc.org)
  • ATP‐γ‐S (EMD Biosciences)
  • Cyclin A‐CDK2 (New England Biolabs)
  • 0.5 M EDTA, pH 8 (MediaTech)
  • Acetonitrile (VWR)
  • 0.5 µg/µl trypsin (sequencing grade, Promega)
  • 0.1% (v/v) formic acid (store in glass bottle at room temperature indefinitely)
  • Disulfide beads: thiopropyl‐Sepharose 6B (GE Healthcare)
  • 20 mM NaOH
  • 0.1% (v/v) formic acid/50% (v/v) acetonitrile (store in glass bottle at room temperature indefinitely)
  • α‐cyano‐4‐hydroxycinnamic acid (Agilent Technologies; also see Jiménez et al., )
  • 1.7‐ml microcentrifuge tubes
  • 0.6 µl C18 ZipTips (Millipore)
  • 4700 Proteomics Analyzer (Applied Biosystems)
  • Additional reagents and equipment for preparing samples for MALDI mass spectrometry (Jiménez et al., ) and MALDI‐TOF mass spectrometry (Henzel and Stults, )
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Allen, J.J., Li, M., Brinkworth, C.S., Paulson, J.L., Wang, D., Hubner, A., Chou, W.H., Davis, R.J., Burlingame, A.L., Messing, R.O., Katayama, C.D., Hedrick, S.M., and Shokat, K.M. 2007. A semisynthetic epitope for kinase substrates. Nat. Methods 4:511‐516.
   Blethrow, J., Zhang, C., Shokat, K.M., and Weiss, E.L. 2004. Design and use of analog‐sensitive protein kinases. Curr. Protoc. Mol. Biol. 66:18.11.11‐18.11.19.
   Blethrow, J.D., Glavy, J.S., Morgan, D.O., and Shokat, K.M. 2008. Covalent capture of kinase‐specific phosphopeptides reveals Cdk1‐cyclin B substrates. Proc. Natl. Acad. Sci. U.S.A. 105:1442‐1447.
   Bond, J.S. 1989. Commercially available proteases. Appendix II in Proteolytic Enzymes, A Practical Protein Chemistry (R.J. Beynon and J.S. Bond, eds.) p. 240. IRL Press, Oxford.
   Buzko, O. and Shokat, K.M. 2002. A kinase sequence database: Sequence alignments and family assignment. Bioinformatics 18:1274‐1275.
   Chi, Y., Welcker, M., Hizli, A.A., Posakony, J.J., Aebersold, R., and Clurman, B.E. 2008. Identification of CDK2 substrates in human cell lysates. Genome Biol. 9:R149.
   Deutsch, E.W., Mendoza, L., Shteynberg, D., Farrah, T., Lam, H., Tasman, N., Sun, Z., Nilsson, E., Pratt, B., Prazen, B., Eng, J.K., Martin, D.B., Nesvizhskii, A.I., and Aebersold, R. 2010. A guided tour of the Trans‐Proteomic Pipeline. Proteomics 10:1150‐1159.
   Henzel, W.J. and Stults, J.T. 1996. Matrix‐assisted laser desorption/ionization time‐of‐flight mass analysis of peptides. Curr. Protoc. Protein Sci. 4:16.2.1‐16.2.11.
   Hertz, N.T., Wang, B.T., Allen, J.J., Zhang, C., Dar, A.C., Burlingame, A.L., and Shokat, K.M. 2010. Chemical genetic approach for kinase‐substrate mapping by covalent capture of thiophosphopeptides and analysis by mass spectrometry. Curr. Protoc. Chem. Biol. 2:15‐36.
   Holt, L.J., Tuch, B.B., Villen, J., Johnson, A.D., Gygi, S.P., and Morgan, D.O. 2009. Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution. Science 325:1682‐1686.
   Jiménez, C., Huang, L., Qiu, Y. and Burlingame, A. 1998. Sample preparation for MALDI mass analysis of peptides and proteins. Curr. Protoc. Protein Sci.. 14:16.3.1–16.3.6.
   Kapp, E. and Schutz, F. 2007. Overview of tandem mass spectrometry (MS/MS) database search algorithms. Curr. Protoc. Protein Sci. 49:25.22.21‐25.22.19.
   Koch, A. and Hauf, S. 2010. Strategies for the identification of kinase substrates using analog‐sensitive kinases. Eur. J. Cell Biol. 89:184‐193.
   Liu, Y., Shah, K., Yang, F., Witucki, L., and Shokat, K.M. 1998. A molecular gate which controls unnatural ATP analogue recognition by the tyrosine kinase v‐Src. Bioorg. Med. Chem. 6:1219‐1226.
   Parker, L.L., Schilling, A.B., Kron, S.J., and Kent, S.B. 2005. Optimizing thiophosphorylation in the presence of competing phosphorylation with MALDI‐TOF‐MS detection. J. Proteome Res. 4:1863‐1866.
   Phelan, M.C. 2007. Basic techniques in mammalian cell tissue culture. Curr. Protoc. Cell Biol. 36:1.1.1–1.1.18.
   Qin, X.Q., Chittenden, T., Livingston, D.M., and Kaelin, W.G. Jr. 1992. Identification of a growth suppression domain within the retinoblastoma gene product. Genes Dev. 6:953‐964.
   Shah, K. and Shokat, K.M. 2003. A chemical genetic approach for the identification of direct substrates of protein kinases. Methods Mol. Biol. 233:253‐271.
   Shah, K., Liu, Y., Deirmengian, C., and Shokat, K.M. 1997. Engineering unnatural nucleotide specificity for Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates. Proc. Natl. Acad. Sci. U.S.A. 94:3565‐3570.
   Williams, A. and Frasca, V. 2001. Ion‐exchange chromatography. Curr. Protoc. Protein Sci. 15:8.2.1‐8.2.30.
   Yi, E.C., Lee, H., Aebersold, R., and Goodlett, D.R. 2003. A microcapillary trap cartridge‐microcapillary high‐performance liquid chromatography electrospray ionization emitter device capable of peptide tandem mass spectrometry at the attomole level on an ion trap mass spectrometer with automated routine operation. Rapid Commun. Mass Spectrom. 17:2093‐2098.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library