Protein phosphorylation analysis in archival clinical cancer samples by shotgun and targeted proteomics approaches

Protein phosphorylation affects most eukaryotic cellular processes and its deregulation is considered a hallmark of cancer and other diseases. Phosphoproteomics may enable monitoring of altered signaling pathways as a means of stratifying tumors and facilitating the discovery of new drugs. Unfortuna...

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Bibliographic Details
Published in:Molecular bioSystems 2011-08, Vol.7 (8), p.2368-2374
Main Authors: Gámez-Pozo, Angelo, Sánchez-Navarro, Iker, Calvo, Enrique, Díaz, Esther, Miguel-Martín, María, López, Rocío, Agulló, Teresa, Camafeita, Emilio, Espinosa, Enrique, López, Juan Antonio, Nistal, Manuel, Vara, Juan Ángel Fresno
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Chromatography, Affinity
Electrophoresis, Polyacrylamide Gel
Histocytochemistry
Humans
Molecular Sequence Data
Neoplasm Proteins - analysis
Neoplasm Proteins - metabolism
Neoplasms - chemistry
Neoplasms - metabolism
Peptide Mapping - methods
Phosphoproteins - analysis
Phosphoproteins - metabolism
Proteomics - methods
Tandem Mass Spectrometry
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Summary:Protein phosphorylation affects most eukaryotic cellular processes and its deregulation is considered a hallmark of cancer and other diseases. 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. Here we report phosphopeptide analysis in human archival formalin-fixed, paraffin-embedded (FFPE) cancer samples based on immobilized metal affinity chromatography followed by liquid chromatography coupled with tandem mass spectrometry and selected reaction monitoring techniques. Our results indicate the equivalence of detectable phosphorylation rates in archival FFPE and fresh frozen tissues. Moreover, we demonstrate the applicability of targeted assays for phosphopeptide analysis in clinical archival FFPE samples, using an experimental workflow suitable for processing and analyzing large sample series. This work paves the way for the application of shotgun and targeted phosphoproteomics approaches in clinically relevant studies using archival clinical samples.
ISSN:1742-206X
1742-2051
DOI:10.1039/c1mb05113j