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

  1. Angelo Gámez-Pozo1,
  2. Iker Sánchez-Navarro1,
  3. Nuria Ibarz Ferrer2,
  4. Fernando García Martínez2,
  5. Keith Ashman2,3,
  6. Juan Ángel Fresno Vara1

Published Online: 1 JUN 2012

DOI: 10.1002/9780470559277.ch110242

Current Protocols in Chemical Biology

Current Protocols in Chemical Biology

How to Cite

Gámez-Pozo, A., Sánchez-Navarro, I., Ibarz Ferrer, N., García Martínez, F., Ashman, K. and Fresno Vara, J. Á. 2012. High-Throughput Phosphoproteomics from Formalin-Fixed, Paraffin-Embedded Tissues. Current Protocols in Chemical Biology. 4:161–175.

Author Information

  1. 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. 2

    Unidad de Proteómica, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain

  3. 3

    Clinical Applications Development, UQCCR, University of Queensland, Australia

Publication History

  1. Published Online: 1 JUN 2012
  2. Published Print: JUN 2012


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.


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