Isolation of Phosphopeptides by Immobilized Metal Ion Affinity Chromatography

Thomas Nühse1, Kebing Yu2, Arthur Salomon2

1 University of Manchester, Manchester, 2 Brown University, Providence, Rhode Island
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.13
DOI:  10.1002/0471142727.mb1813s77
Online Posting Date:  January, 2007
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Abstract

The identification of protein phosphorylation sites from cell‐derived proteins is crucial to the understanding of signal transduction pathways. While determining the modified sites on individual proteins can present a significant challenge, recent progress in the rapid, large‐scale identification of phosphopeptides from complex protein mixtures by combinations of affinity chromatography and mass spectrometry provides a powerful tool to decipher the phosphoproteome. A set of protocols is described for sample preparation, fractionation, immobilized metal ion affinity chromatography (IMAC), and mass spectrometric analysis of phosphorylation sites. Parts of the protocols can be combined in different ways to adapt to sample amounts, complexity, and available equipment. Up to thousands of unique phosphopeptides can be sequenced from a single sample in a day, revealing a unique snapshot of global cellular phosphorylation sites on proteins and facilitating in‐depth study of the identified phosphoproteins.

Keywords: phosphorylation; phosphopeptide; mass spectrometry; proteomics; signal transduction

     
 
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Table of Contents

  • Basic Protocol 1: Manual Peptide Fractionation by Cation‐Exchange Chromatography and IMAC
  • Alternate Protocol 1: Manual Peptide Fractionation by Anion‐Exchange Chromatography and IMAC
  • Support Protocol 1: Manual Chromatography with Microcolumns
  • Basic Protocol 2: Enrichment of Phosphopeptides Through Methyl Ester Formation and IMAC
  • Support Protocol 2: Construction of Columns for IMAC and Reversed‐Phase Separation of Methyl Ester–Derivitized Peptides
  • Basic Protocol 3: Enrichment of Tyrosine‐Phosphorylated Peptides via Peptide Immunoprecipitation and IMAC
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Manual Peptide Fractionation by Cation‐Exchange Chromatography and IMAC

  Materials
  • Protein sample (100 to 500 µg)
  • 100 mM sodium carbonate (Na 2CO 3)
  • 50 and 500 mM triethylammonium bicarbonate (TEAB) in ddH 2O, diluted from 1 M stock solution (Sigma)
  • Sequencing‐grade modified trypsin
  • 2.5% (v/v) formic acid/30% (v/v) acetonitrile
  • Cation‐exchange cartridge (e.g., Optimize Techologies Opti‐Lynx or Applied Biosystems iTRAQ), with holder and syringe adapter
  • SCX start buffer (see recipe)
  • SCX step elution buffers (10, 20, 30, 40, 60, and 100 mM salt; see recipe)
  • SCX cleaning buffer (see recipe)
  • Sodium azide, optional
  • Phos‐Select IMAC resin (Sigma)
  • IMAC load/wash buffer (see recipe)
  • IMAC elution buffer (see recipe)
  • 10% (v/v) formic acid
  • 11 × 34–mm polycarbonate ultracentrifuge tubes
  • Ultracentrifuge (e.g., Beckman Optima tabletop ultracentrifuge with a TLA120 rotor)
  • 37°C water bath
  • Lyophilizer or Speedvac evaporator
  • 1.5‐ and 2‐ml microcentrifuge tubes
  • End‐over‐end rotator
  • Fritted spin columns (e.g., Mobicols, MoBiTec; Handee Mini Spin columns, Pierce)

Alternate Protocol 1: Manual Peptide Fractionation by Anion‐Exchange Chromatography and IMAC

  • POROS HQ strong anion‐exchange resin
  • SAX start buffer (see recipe)
  • SAX step buffer (see recipe)
  • SAX step elution buffers (50, 100, 200, and 500 mM; see recipe)
  • GELoader tips (Eppendorf)

Support Protocol 1: Manual Chromatography with Microcolumns

  Materials
  • Protein sample (1 × 108 cells or ∼100 fmol purified protein)
  • Cell lysis buffer A (see recipe), optional
  • Immunoprecipitation resin: anti‐phosphotyrosine agarose (e.g., clone PT66, Sigma), optional
  • Immunoprecipitation wash buffer (see recipe), optional
  • Trypsin digestion buffer with urea (see recipe)
  • Milli‐Q water
  • Sequencing‐grade modified trypsin (Promega)
  • Dialysis buffer (see recipe), optional
  • Trizol LS (Invitrogen), optional
  • DC protein assay kit (Bio‐Rad) containing:
    • Alkaline copper tartrate solution
    • Folin reagent
    • Surfactant solution
    • Bovine gamma‐globulin standard
  • Angiotensin II phosphate (EMD Biosciences) or other synthetic phosphopeptide
  • 1 M HCl
  • Reversed‐phase loading buffer: 0.1% acetic acid in Milli‐Q water
  • Reversed‐phase elution buffer (see recipe)
  • Macrotrap wash buffer 1: 20:80 (v/v) dimethylsulfoxide (DMSO)/methanol
  • Macrotrap wash buffer 2: 45:45:10 (v/v/v) isopropanol/water/formic acid
  • Methylation solution (see recipe)
  • Nitrogen source
  • Fe(III)‐activated IMAC column (see protocol 5)
  • IMAC preparation buffer 1: 50 mM EDTA, pH 8
  • IMAC preparation buffer 2: 100 mM FeCl 3 (99.99+%, Sigma)
  • Desalting column (see protocol 5)
  • HPLC buffer A (IMAC): 0.01% acetic acid in Milli‐Q ddH 2O
  • HPLC buffer B (IMAC): 0.01% acetic acid in acetonitrile (99.8%; HPLC grade)
  • IMAC wash buffer (see recipe)
  • Precolumn (see protocol 5)
  • IMAC elution buffer: 25 mM Na 2HPO 4, pH 9.0
  • Analytical column (see protocol 5)
  • HPLC buffer C (reversed‐phase): 0.1 M acetic acid in Milli‐Q ddH 2O
  • HPLC buffer D (reversed‐phase): 0.1 M acetic acid in acetonitrile (99.8%; HPLC grade)
  • 1‐ml syringes and 27‐G needles
  • 50‐ml conical tubes
  • 1.5‐ml microcentrifuge tubes
  • 95°C heating block
  • 0.45‐µm, 4‐mm syringe filters (e.g., Millipore Millex), optional
  • 200‐ and 1000‐µl pipets (Rainin)
  • 10,000 MWCO Slide‐A‐Lyzer (Pierce), optional
  • Reversed‐phase C18 peptide macrotrap (Michrom Bioresources)
  • 1.5‐ml screw‐top microcentrifuge tubes (Sarstedt)
  • Speedvac evaporator (Thermo Savant)
  • Inline microfilters (Upchurch Scientific)
  • HPLC pump (e.g., Agilent 1100 Quaternary Pump)
  • Fused silica capillaries (360‐µm o.d. × 50‐, 75‐, and 200‐µm i.d.; Polymicro Technologies)
  • Microtee (Upchurch Scientific)
  • Laser Puller P2000 (Sutter Instruments), optional
  • Mass spectrometer (e.g., LTQ‐FTICR and LTQ available from Thermo)
  • Valco trap holder (Michrom Bioresources)
  • Multiple‐syringe infusion pump
  • 500‐µl Hamilton syringe
  • Platinum wire (Sigma)
NOTE: All sample preparation procedures involving intact proteins are carried out at 4°C unless otherwise specified.

Basic Protocol 2: Enrichment of Phosphopeptides Through Methyl Ester Formation and IMAC

  Materials
  • Anti‐phosphotyrosine antibody (clone PTyr100, Cell Signalling Technology)
  • Antibody coupling buffer: 20 mM sodium phosphate, pH 7.0
  • Protein G–agarose beads (Roche)
  • Protein sample (3 × 107 cells or ∼100 fmol purified protein)
  • Cell lysis buffer B (see recipe)
  • Dithiothreitol (DTT)
  • Iodoacetamide
  • 100 mM ammonia bicarbonate, pH 8.9
  • DC protein assay kit (Bio‐Rad) containing:
    • Alkaline copper tartrate solution
    • Folin reagent
    • Surfactant solution
    • Bovine gamma‐globulin standard
  • Affinity purified, TPCK‐treated trypsin (Promega)
  • Concentrated HCl
  • Sep‐Pak C18 plus column (Waters)
  • 100% acetonitrile
  • Sep‐Pak loading buffer: 0.1% acetic acid in Milli‐Q water
  • Sep‐Pak elution buffer: 25% acetonitrile/0.1% acetic acid in Milli‐Q water
  • Peptide immunoprecipitation (IP) buffer (see recipe), 4°C
  • Synthetic phosphopeptide external standard
  • pTyr‐100 immobilized antibody
  • Peptide IP wash buffer 1 (see recipe)
  • Peptide IP wash buffer 2 (see recipe)
  • Peptide IP elution buffer: 100 mM glycine, pH 2.5
  • Tyrosine‐phosphorylated standard peptides (e.g., angiotensin II phosphate, EMD Biosciences; or various pTyr peptides, Anaspec)
  • End‐over‐end rotator, 4°C
  • 56°C water bath
  • 15‐ml conical tubes
  • 1.5‐ml screw‐top microcentrifuge tubes (Sarstedt)
  • 18‐ and 27‐G needles
  • Speedvac evaporator (Thermo Savant)
  • Additional reagents and equipment for IMAC enrichment and MS analysis (see protocol 4)
NOTE: All sample preparation procedures involving intact proteins are carried out at 4°C unless otherwise specified.
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Figures

Videos

Literature Cited

Literature Cited
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