Determining In Vivo Phosphorylation Sites Using Mass Spectrometry

Susanne B. Breitkopf1, John M. Asara2

1 Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts, 2 Department of Medicine, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.19
DOI:  10.1002/0471142727.mb1819s98
Online Posting Date:  April, 2012
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Abstract

Phosphorylation is the most studied protein post‐translational modification (PTM) in biological systems, since it controls cell growth, proliferation, survival, and other processes. High‐resolution/high mass accuracy mass spectrometers are used to identify protein phosphorylation sites due to their speed, sensitivity, selectivity, and throughput. The protocols described here focus on two common strategies: (1) identifying phosphorylation sites from individual proteins and small protein complexes, and (2) identifying global phosphorylation sites from whole‐cell and tissue extracts. For the first, endogenous or epitope‐tagged proteins are typically immunopurified from cell lysates, purified via gel electrophoresis or precipitation, and enzymatically digested into peptides. Samples can be optionally enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) and then analyzed by microcapillary liquid chromatography/tandem mass spectrometry (LC‐MS/MS). Global phosphorylation site analyses that capture pSer/pThr/pTyr sites from biological sources sites are more resource and time consuming and involve digesting the whole‐cell lysate, followed by peptide fractionation by strong cation‐exchange chromatography, phosphopeptide enrichment by IMAC or TiO2, and LC‐MS/MS. Alternatively, the protein lysate can be fractionated by SDS‐PAGE, followed by digestion, phosphopeptide enrichment, and LC‐MS/MS. One can also immunoprecipitate only phosphotyrosine peptides using a pTyr antibody followed by LC‐MS/MS. Curr. Protoc. Mol. Biol. 98:18.19.1‐18.19.27. © 2012 by John Wiley & Sons, Inc.

Keywords: phosphorylation; mass spectrometry; LC‐MS/MS; SCX; IMAC; immunoprecipitation; SDS‐PAGE; nano‐LC; phosphoproteomics

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Single‐Protein (Protein Complex) Phosphorylation Site Mapping
  • Alternate Protocol 1: Acetone Precipitation of Protein Samples (for IP Elution with Peptide or Small Molecule)
  • Support Protocol 1: Phosphopeptide Enrichment Using Protea Biosciences TiO2 Spin Tips
  • Support Protocol 2: Phosphopeptide Enrichment Using the Phos‐Trap TiO2 Phosphopeptide Enrichment Kit
  • Basic Protocol 2: Global Phosphorylation Analysis (Ser/Thr/Tyr)
  • Alternate Protocol 2: SDS‐PAGE Protein Fractionation of Whole‐Cell Extracts
  • Basic Protocol 3: Phosphotyrosine (pTyr) Site Identification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Single‐Protein (Protein Complex) Phosphorylation Site Mapping

  Materials
  • Cells/tissue source (e.g., cell line, tumor, extracted bodily tissue)
  • Lysis/IP buffer (see recipe)
  • Liquid nitrogen (−196°C)
  • Bradford protein assay kit (BioRad; also see unit 10.1)
  • Antibody targeting protein of interest
  • Protein A (GE Healthcare, cat. no. 17‐0963‐03) or G (GE Healthcare, cat. no. 17‐0618‐02) agarose beads
  • 1× SDS sample buffer (see recipe)
  • SDS‐PAGE Tris‐glycine polyacrylamide minigels 10‐well, 1‐mm (10% fixed or 4% to 20% gradient; Lonza, cat. no. 58511)
  • Tris‐glycine SDS running buffer (1× formulation: 25 mM Tris⋅Cl, 192 mM glycine, 0.1% SDS, pH 8.3)
  • Coomassie blue stain (15% methanol, 10% acetic acid, 2 g Coomassie Brilliant Blue)
  • Coomassie destain (15% methanol, 10% glacial acetic acid)
  • 50% (v/v) acetonitrile/H 2O (LC‐MS grade)
  • 10 mM dithiothreitol (DTT) in 100 mM ammonium bicarbonate (NH 4HCO 3)
  • 100% acetonitrile
  • 55 mM iodoacetamide (IAA) in 100 mM NH 4HCO 3
  • 100 mM, 50 mM and 20 mM NH 4HCO 3
  • 25 ng/µl trypsin (see recipe)
  • 2% (v/v) formic acid/40% (v/v) acetonitrile
  • HPLC buffer A: 99% (v/v) H 2O, 0.9% acetonitrile, 0.1% formic acid
  • HPLC buffer B: 100% acetonitrile
  • Stainless steel mortar with ceramic pestle
  • Platform rocker with circular motion
  • 15‐ml conical polypropylene centrifuge tubes
  • Refrigerated centrifuge and microcentrifuge
  • End‐over‐end rotator
  • 56° and 95°C heat block or water bath
  • Minigel SDS‐PAGE apparatus (BioRad or other vendor) with power supply
  • 37°C shaking incubator
  • Nanoflow HPLC: ThermoFisherScientific EASY‐nLC, Waters NanoAcquity, Eksigent NanoLC Ultra (http://www.eksigent.com/), Bruker Nanoflow‐LC, or equivalent
  • Pico‐Frit packed C 18 columns: 75 µm ID × 15 cm length (New Objective, PF7515‐150H002‐3P; http://www.newobjective.com/)
  • High resolution/high mass accuracy mass spectrometer: ThermoFisherScientific (LTQ‐Orbitrap XL, Velos Pro Orbitrap, Velos Elite Orbitrap, qExactive), Waters Xevo, AB/Sciex 5600, Agilent QTOF 6500 series, Bruker microTOF, or equivalent
  • Database search engine software: e.g., Sequest (ThermoFisherScientific) or Mascot (Matrix Science; http://www.matrixscience.com/)
  • Proteomics Browser Software (ThermoFisherScientific or http://www.mcb.harvard.edu/microchem/)
  • Scaffold PTM Software (Proteome Software, Inc.; http://www.proteomesoftware.com/)
  • Additional reagents and equipment for protein assay (unit 10.1), SDS‐PAGE (unit 10.2), and staining of gels (unit 10.6),

Alternate Protocol 1: Acetone Precipitation of Protein Samples (for IP Elution with Peptide or Small Molecule)

  • Acetone
  • Protein sample
  • 1 µg/µl TPCK‐modified trypsin prepared in 50 mM acetic acid
  • 5% (w/v) trifluoroacetic acid (TFA)
  • ZipTip binding and wash buffer: 0.1% (w/v) trifluoroacetic acid (TFA)
  • ZipTip elution buffer: 0.1% (w/v) trifluoroacetic acid/40% acetonitrile
  • Acetone‐compatible tubes (polypropylene only)
  • pH paper
  • C 18 ZipTips (Millipore, cat. no. ZTC18S096)
  • 0.5‐ml microcentrifuge tubes or 12 × 32 mm autosampler vials (National Scientific, cat. no. C4000‐87)

Support Protocol 1: Phosphopeptide Enrichment Using Protea Biosciences TiO2 Spin Tips

  • Peptide sample
  • Protea TiO 2 SpinTips Sample Prep Kit (Protea Biosciences) including:
    • SpinTip adapers
    • TiO 2 Reconstitution and Wash Solution 1
    • TiO 2 Elution Solution
  • 2‐ml microcentrifuge tubes

Support Protocol 2: Phosphopeptide Enrichment Using the Phos‐Trap TiO2 Phosphopeptide Enrichment Kit

  Materials
  • Peptide sample
  • Phos‐Trap TiO 2 phosphopeptide enrichment kit (PerkinElmer) including:
    • Magnetic beads
    • Binding buffer
    • Wash buffer
    • Elution buffer
  • Magnet (bench top, for microcentrifuge tubes)
  • 12 × 32 mm autosampler vials (National Scientific, cat. no. C4000‐87)

Basic Protocol 2: Global Phosphorylation Analysis (Ser/Thr/Tyr)

  Materials
  • Cells of interest, in log phase
  • Urea lysis buffer (see recipe)
  • Liquid nitrogen (−196°C)
  • Bradford protein assay kit (BioRad; also see unit 10.1)
  • 45 mM DTT: mix 180 µl of 1.25 M DTT (19.25 g/100 ml) with 5 ml HPLC‐grade water; add to sample at ∼1/10 dilution (e.g., 0.5 ml DTT per 5‐ml sample)
  • 110 mM IAA: Dissolve 209 mg iodoacetamide (IAA) in 10 ml HPLC‐grade water; add to sample at ∼1/10 dilution
  • Sequencing grade modified trypsin (100 µg/vial; Worthington, cat. no. LS02122)
  • 50 mM ammonium bicarbonate (NH 4 HCO 3)
  • 20%, 10%, 1%, 0.1% (v/v) trifluoroacetic acid (TFA)
  • 0.1% TFA/40% acetonitrile
  • SCX buffer A: 7 mM KH 2PO 4, pH 2.65 in 30% (v/v) acetonitrile
  • SCX buffer B: 7 mM KH 2PO 4 /350 mM KCl, pH 2.65 in 30% (v/v) acetonitrile
  • IMAC (PHOS‐Select Iron affinity gel, Sigma, cat. no. P9740)
  • IMAC binding buffer (40% acetonitrile (v/v), 25 mM formic acid, H 2O)
  • IMAC elution buffer A (50 mM K 2HPO 4/NH 4OH, pH 10.0)
  • IMAC elution buffer B (500 mM K 2HPO 4, pH 7)
  • Methanol
  • 40% (v/v) acetonitrile/0.5% acetic acid
  • 1% (v/v) formic acid
  • Stainless steel mortar with ceramic pestle
  • Bath sonicator
  • Centrifuge
  • 56°C water bath or heat block
  • pH paper
  • Sep‐Pak C 18 cartridges 6‐cc/500 mg (Waters, WAT036790) for whole digested lysate
  • Strong cation exchange (SCX) column: PolySULFOETHYL A 250 × 9.4 mm; 5 µm pore size; 200 Å (PolyLC, 259‐SE0502)
  • Nanoflow HPLC: ThermoFisherScientific EASY‐nLC, Waters NanoAcquity, Eksigent NanoLC Ultra (http://www.eksigent.com/), Bruker Nanoflow‐LC, or equivalent
  • 1‐ml sample loop
  • High‐flow‐rate HPLC (offline SCX fractionation): e.g., Agilent 1200, Michrom Paradigm MG4 (Bruker), Shimadzu Prominence
  • 15‐ml conical centrifuge tubes
  • Sep‐Pak C 18 cartridges 3‐cc/50 mg (Waters, WAT054960) for fractionated sample (after SCX); one cartridge for each fraction (12 SCX fractions = 12 Sep‐Pak 3‐cc/50‐mg cartridges)
  • 0.5‐ml microcentrifuge tubes
  • Empore 3M C 18 material (http://www.shop3m.com/)
  • Cutter device (Hamilton, Needle Kel‐F hub (KF), point style 3, gauge 16, cat. no. 90516; plunger assembly N, RN, LT, LTN for model 1702 (25 µl), cat. no. 1122‐01
  • 200‐µl pipet tips for StageTip preparation
  • 12 × 32 mm autosampler vials (National Scientific, cat. no. C4000‐87)
  • High‐resolution/high mass accuracy mass spectrometer: ThermoFisherScientific (LTQ‐Orbitrap XL, Velos Pro Orbitrap, Velos Elite Orbitrap, qExactive), Waters Xevo, AB/Sciex 5600, Agilent QTOF 6500 series, Bruker microTOF, or equivalent
  • Additional reagents and equipment for protein assay (unit 10.1)
NOTE: For SCX buffers, organic solvents affect the pH reading. The pH adjustments for SCX buffers A and B (see above) should be performed before the addition of acetonitrile.

Alternate Protocol 2: SDS‐PAGE Protein Fractionation of Whole‐Cell Extracts

  Materials
  • Digested and purified sample pellet ( protocol 5, step 24)
  • Lysis/IP buffer (see recipe)
  • 1 M Tris base (pH not adjusted)
  • Phosphotyrosine P‐Tyr‐100 mouse antibody (mAb), Sepharose conjugated (Cell Signaling Technology, cat. no. 9419)
  • P‐Tyr‐100 elution buffer: 0.15% (v/v) trifluoroacetic acid (TFA)
  • 100% acetonitrile
  • 0.1% (v/v) trifluoroacetic acid (TFA)/40% acetonitrile
  • 0.1% (v/v) trifluoroacetic acid
  • Bath sonicator
  • End‐over‐end rotator
  • Gel‐loading pipet tips
  • Refrigerated centrifuge
  • C 18 ZipTips (Millipore, cat. no. ZTC18S096)
  • Nanoflow HPLC: ThermoFisherScientific EASY‐nLC, Waters NanoAcquity, Eksigent NanoLC Ultra (http://www.eksigent.com/), Bruker Nanoflow‐LC, or equivalent
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Figures

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Literature Cited

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