High‐Throughput Phosphoproteomics from Formalin‐Fixed, Paraffin‐Embedded Tissues

Angelo Gámez‐Pozo1, Iker Sánchez‐Navarro1, Nuria Ibarz Ferrer2, Fernando García Martínez2, Keith Ashman3, Juan Ángel Fresno Vara1

1 Laboratorio de Oncología y Patología Molecular, Instituto de Genética Médica y Molecular‐INGEMM, Instituto de Investigación Hospital Universitario La Paz‐IdiPAZ, Madrid, Spain, 2 Unidad de Proteómica, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain, 3 Clinical Applications Development, UQCCR, University of Queensland, Australia
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110242
Online Posting Date:  June, 2012
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Abstract

Liquid chromatography coupled with tandem mass spectrometry–based high‐throughput proteomics allows detection and characterization of thousands of peptides and their post‐translational modifications in a single sample. Protein phosphorylation affects most eukaryotic cellular processes, and its deregulation is considered a hallmark of cancer and other diseases. High‐throughput phosphoproteomics may enable monitoring of altered signaling pathways as a means of stratifying tumors and facilitating the discovery of new drugs. Unfortunately, the development of molecular tests for clinical use is constrained by the limited availability of fresh frozen, clinically annotated samples, and protocols that allow the use of human archival formalin‐fixed, paraffin‐embedded samples are required. The protocols in this article describe a global procedure for evaluating hundreds of protein phosphorylation sites in protein extracts obtained from formalin‐fixed, paraffin‐embedded tissues. Curr. Protoc. Chem. Biol. 4:161‐175 © 2012 by John Wiley & Sons, Inc.

Keywords: FFPE samples; phosphoproteomics; biomarkers; immobilized metal ion affinity chromatography; mass spectrometry

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Protein Extraction from FFPE Tissues
  • Basic Protocol 2: Phosphopeptide Enrichment by Affinity Chromatography
  • Basic Protocol 3: LC‐ESI‐MS/MS Analysis of Phosphopeptides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Protein Extraction from FFPE Tissues

  Materials
  • FFPE block
  • 100% xylene (ACS grade or higher quality)
  • 100% ethanol (ACS grade or higher quality)
  • Protein extraction buffer (see recipe)
  • BCA Protein Assay Kit (Pierce, cat. no. 23225)
  • Microtome for tissue sectioning (e.g., Microm HM350 S; http://www.microm.de/)
  • Shaker‐incubators with heat blocks capable of incubating at 99°C and shakers capable of shaking at 1000 rpm
NOTE: Plastics used during handling of samples can introduce contaminants that interfere with MS analysis and result in sample loss from nonspecific adsorption. Use high‐quality, low‐binding plastic material.

Basic Protocol 2: Phosphopeptide Enrichment by Affinity Chromatography

  Materials
  • Protein extracts of FFPE tissue samples (from protocol 1; typically containing 500 µg of protein)
  • 50 mM ammonium bicarbonate (see recipe)
  • 1 mg/ml Trypsin Gold (see recipe)
  • Trifluoroacetic acid (TFA; 1‐ml ampules; Pierce, cat. no. 28904)
  • Mass spectrometry‐grade acetonitrile, (e.g., acetonitrile LC‐MS CHROMASOLV; Sigma, cat. no 34967)
  • Fe‐NTA buffer (see recipe)
  • Mass spectrometry‐grade water (e.g., Water LC‐MS CHROMASOLV; Fluka, cat. no. 39253)
  • Acetic acid for LC‐MS (Fluka, cat. no 49199)
  • Pierce Fe‐NTA Phosphopeptide Enrichment Kit (Pierce, cat. no. 88300)
  • Ammonium hydroxide solution, ∼ 25% in H 2O (Fluka, cat. no. 44273)
  • Elution buffer (see recipe)
  • 0.5% (v/v) formic acid (Fluka, cat. no. 56302)
  • 1.5‐ml low‐bind microcentrifuge tubes
  • Graphite spin columns (Pierce, cat. no. 88302)
  • Shaker‐incubators with heat blocks capable of incubating at 99°C and shakers capable of shaking at 1000 rpm
  • Vacuum evaporator
  • Bath sonicator

Basic Protocol 3: LC‐ESI‐MS/MS Analysis of Phosphopeptides

  Materials
  • Sample (from protocol 2)
  • Buffer A: LC‐MS grade water containing 0.1% (v/v) formic acid; dissolve 1‐ml ampule (Fluka, cat. no. 56302) in 999 ml water
  • Buffer B: LC‐MS grade acetonitrile containing 0.1% (v/v) formic acid; dissolve 1 ml ampule (Fluka, cat. no. 56302) in 999 ml acetonitrile
  • 250‐µl polypropylene HPLC vials (Fisher Scientific)
  • Nano‐LC equipped with thermostatic autosampler (e.g., NanoLC‐Ultra 1D plus; Eksigent, http://www.eksigent.com/)
  • Trapping column: Reprosil C18 AQ 5 µm 120A 0.3 × 10 mm (SGE Analytical, http://www.sge.com/)
  • Analytical column: Reprosil‐Pur C18, 3 µm, 0.075 × 200 mm (Dr. Maisch, GmbH, http://www.dr‐maisch.com/ )
  • Nano‐electrospray source (e.g., Nano ES ion source, Proxeon Biosystems, http://www.proxeon.com)
  • Stainless steel emitter (Proxeon ES502, O.D 150 µm, I.D. 30 µm; http://www.proxeon.com)
  • Fused silica capillary (Polymicro Technologies, http://www.polymicro.com/); 360‐µm O.D. and 20, 50, or 75 µl I.D.
  • LTQ‐Orbitrap Velos mass spectrometer (Thermo Scientific)
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Figures

Videos

Literature Cited

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Key References
   Ye et al., 2010. See above.
  This recent reference describes the Fe‐NTA protocol. This publication also describes the comparison of three widely used IMAC materials under three different conditions and demonstrates that Fe(III)‐nitrilotriacetic acid (NTA) IMAC resin showed superior performance in all tests. There are minor differences between their Fe‐NTA protocol and the description provided within this unit.
   Gamez‐Pozo et al., 2011. See above.
  This paper by our group provides a good introduction to the feasibility of using clinical FFPE samples for phosphoproteomics studies focused on biomarker discovery, as well as an overview of important issues related to the use of FFPE samples in high‐throughput proteomics experiments.
   Nagaraj et al., 2010. See above.
  These works describe the advantages introduced by the use of HCD fragmentation plus Orbitrap detection in phosphoproteomic analyses.
   Kelstrup et al., 2011. See above.
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