Tyrosine Phosphorylation Enrichment and Subsequent Analysis by MALDI‐TOF/TOF MS/MS and LC‐ESI‐IT‐MS/MS

Mark R. Condina1, Manuela Klingler‐Hoffmann2, Peter Hoffmann1

1 Adelaide Proteomics Centre, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia, 2 Chemokine Biology Laboratory, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 13.11
DOI:  10.1002/0471140864.ps1311s62
Online Posting Date:  November, 2010
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Abstract

This unit describes methods to detect, identify, and characterize tyrosine phosphorylation in proteins by mass spectrometry, including sample preparation methods, enrichment strategies using phosphotyrosine‐specific antibodies, and chromatographic separation methods. Curr. Protoc. Protein Sci. 62:13.11.1‐13.11.26. © 2010 by John Wiley & Sons, Inc.

Keywords: ESI‐IT‐MS/MS; liquid chromatography; MALDI; matrix preparation; tyrosine phosphorylation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Pro‐Gp Magnetic Bead Enrichment of Tyrosine Phosphopeptides
  • Basic Protocol 2: Cov‐P Magnetic Bead Enrichment of Tyrosine Phosphopeptides
  • Tyrosine Phosphorylation: Global Enrichment Strategies
  • Basic Protocol 3: Tyrosine Phosphoprotein Enrichment from Cell Lysates Using 4G10 and Protein A Sepharose Column
  • Basic Protocol 4: Tyrosine Phosphopeptide Enrichment from Cell Lysates Using 4G10 and Protein A Sepharose Column
  • Support Protocol 1: Prot‐A 4G10 Column Generation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Pro‐Gp Magnetic Bead Enrichment of Tyrosine Phosphopeptides

  Materials
  • MB‐IAC Prot G magnetic bead kit (Bruker Daltonics, cat. no. 100‐03D; store up to 3 months at 4°C) containing:
    • Magnetic beads protein G
    • MB‐IAC Prot G Ab‐immobilization buffer (IB)
    • MB‐IAC Prot G Ag‐binding buffer (BB)
    • MB‐IAC Prot G Ag‐wash buffer (WB)
  • pTyr‐specific Ab 4G10 (Upstate Biotechnology, cat. no. 05‐321)
  • Protein digests of phosphorylated and non‐phosphorylated peptides to be analyzed
  • 50 mM ammonium bicarbonate, pH ∼7.8 (see recipe), optional
  • 2,5‐Dihydroxybenzoic acid (DHB) dissolved at 10 mg/ml in water or 0.1% trifluoroacetic acid (TFA) with 1% phosphoric acid
  • 0.2‐ml PCR polypropylene tubes (e.g., Sarstedt four‐strip PCR tubes, cat. no. 72.990)
  • Magnet separator for 8‐fold PCR tubes (Bruker Daltonics, cat. no. 65554)
  • Gel loader tips
  • Bench‐top vortex equipment or rotating wheel
  • Bruker AnchorChip targets

Basic Protocol 2: Cov‐P Magnetic Bead Enrichment of Tyrosine Phosphopeptides

  Materials
  • MB‐CovAC‐Select magnetic bead kit (Bruker Daltonics; store up to 3 months at 4°C) containing:
    • Magnetic MB‐CovAC‐Select beads
    • MB‐CovAC‐Select immobilization buffer (IB)
    • MB‐CovAC‐Select binding buffer (BB)
    • MB‐CovAC‐Select wash buffer 1 (WB1)
    • MB‐CovAC‐Select wash buffer 2 (WB2)
    • MB‐CovAC‐Select wash buffer 3 (WB3)
  • 4G10 Ab (Upstate Biotechnology, cat. no. 05‐321)
  • 8% ethanolamine (2‐aminoethanol) solution, pH 8.5 (see recipe)
  • 20% sodium azide solution (see recipe)
  • Protein digests of phosphorylated and non‐phosphorylated peptides
  • 2,5‐Dihydroxybenzoic acid (DHB) dissolved at 10 mg/ml in water or 0.1% trifluoroacetic acid (TFA) with 1% phosphoric acid
  • 50 mM ammonium bicarbonate, pH ∼7.8 (see recipe), optional
  • 2‐ml polypropylene microcentrifuge tubes
  • Magnetic separator for 2‐ml tube (e.g., Qiagen, cat. no. 36910 or Promega, cat. no. Z5332)
  • Gel loader tips
  • Bench‐top vortex equipment or rotating wheel
  • 0.2‐ml PCR polypropylene tubes (e.g., Sarstedt four‐strip PCR tubes, cat. no. 72.990)
  • Magnetic separator for 8‐fold PCR tubes (Bruker Daltonics, cat. no. 65554)
  • MALDI target

Basic Protocol 3: Tyrosine Phosphoprotein Enrichment from Cell Lysates Using 4G10 and Protein A Sepharose Column

  Materials
  • Cells of interest
  • 1× phosphate buffered saline (PBS; appendix 2E), store at 4°C
  • Modified radioimmunoprecipitation (RIPA) buffer or other lysis buffer compatible with affinity purification for cell lysis (with inhibitors; see recipe)
  • Protein concentration determination assay, e.g., EZQ protein quantitation assay (Invitrogen and Molecular Probes, cat. no. R33200; also see unit 3.4
  • Protein A Sepharose beads
  • 4G10 Ab protein A column (see protocol 5)
  • Wash buffer: 50 mM Tris⋅Cl, pH 8.0 with 1 mM Na 3VO 4, 25 mM NaCl (store at room temperature; add NA 3VO 4 just before use)
  • Elution buffer: 150 mM phenylphosphate in wash buffer (prepare fresh)
  • Platform shaker and rotating wheel at 4°C
  • Centrifuge tubes
  • Refrigerated centrifuge
  • 37°C water bath
  • Vacuum concentrator or molecular weight spin filter (e.g., vivaspin with 3‐kDa cut‐off)

Basic Protocol 4: Tyrosine Phosphopeptide Enrichment from Cell Lysates Using 4G10 and Protein A Sepharose Column

  Materials
  • 80% and 100% acetone
  • Modified RIPA buffer (see recipe)
  • Protein concentration determination assay (e.g., EZQ protein quantitation assay, Invitrogen and Molecular Probes, cat. no. R33200; or unit 3.4)
  • Protease (e.g., trypsin)
  • Reducing agent (e.g., dithiothreitol–DTT, Sigma‐Aldrich, cat. no. D0632)
  • Alkylation reagent (e.g., iodoacetamide–IAM, Sigma‐Aldrich, cat. no. I1149)
  • Formic acid
  • 4G10 Ab Protein A column (see protocol 5)
  • Binding buffer: 10 mM ammonium bicarbonate solution (NH 4HCO 3), pH 7.8 (see recipe)
  • 2 N NaOH or 2 N HCl
  • Elution buffer: 150 mM phenylphosphate in binding buffer, prepare fresh
  • Refrigerated centrifuge
  • C 18 columns (e.g., for small samples, C 18 ZipTip, Millipore, cat. no. ZTC18S960; for large samples, SepPak catridges, Millipore, cat. no. WAT054955 or columns—Alltech, cat. no. 28960—that have been packed in‐house using Agilent Zorbax‐C18 packing material, cat. no. 820966‐402)
  • Rotating wheel at 4°C
  • Vacuum concentrator
  • Additional reagents and equipment for sample preparation (see protocol 3)

Support Protocol 1: Prot‐A 4G10 Column Generation

  Materials
  • Protein A Sepharose beads (Prot A fast‐flow Sepharose beads, GE Healthcare, cat. no. 17‐5280‐01)
  • Binding buffer: 50 mM Tris⋅Cl, pH 8.0
  • 4G10 antibody (Upstate Biotechnology, cat. no. 05‐321)
  • 20% sodium azide (NaN 3; see recipe)
  • Poly‐Prep chromatography columns (Bio‐Rad, cat. no. 731‐1550)
  • Micro Bio‐Spin chromatography columns (Bio‐Rad, cat. no. 732‐6204)
  • Rotating wheel
  • Spectrophotometer
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Figures

Videos

Literature Cited

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