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A Combination of Affinity Chromatography, 2D DIGE, and Mass Spectrometry to Analyze the Phosphoproteome of Liver Progenitor Cells

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Liver Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 909))

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Abstract

Reversible protein phosphorylation is a ubiquitous posttranslational modification that regulates cellular signaling pathways in multiple biological processes. A comprehensive analysis of protein phosphorylation patterns can only be achieved by employing different complementary experimental strategies all aiming at selective enrichment of phosphorylated proteins/peptides. In this chapter, we describe a method that utilizes a phosphoprotein affinity chromatography (Qiagen) to isolate intact phosphoproteins. These are subsequently detected by difference in two-dimensional gel electrophoresis and identified by mass spectrometry techniques. Additional experiments using a specific stain for phosphoproteins demonstrated that phosphoprotein affinity column was an effective method for enriching phosphate-containing proteins. Further validating the method, this workflow was applied to probe changes in the activation patterns of intermediates involved in different signaling pathways, such as NDRG1 and stathmin, in liver progenitor cells (MLP-29) upon proteasome inhibition.

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Acknowledgements

This laboratory is member of the National Institute of Proteomics Facilities, Proteored. Parts of the work described here were supported by the agreement between FIMA and the “UTE project CIMA”; grants Plan Nacional I  +  D  +  I SAF2008-0154 from Ministerio de Ciencia e Innovación to FJC; ISCIII-RETIC RD06/0020 to MAA and FJC. We like to acknowledge technical contributions from Carmen Miqueo, María I. Mora, and Manuela Molina from Center for Applied Medical Research (CIMA, University of Navarra).

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Authors and Affiliations

  1. Proteomics Unit, Biomedical Research Center, Navarra Health Service, Pamplona, Spain

    Enrique Santamaría & Joaquín Fernández-Irigoyen

  2. Division of Hepatology and Gene Therapy, Proteomics Unit, Centre for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain

    Virginia Sánchez-Quiles & Fernando J. Corrales

Authors
  1. Enrique Santamaría
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  2. Virginia Sánchez-Quiles
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  3. Joaquín Fernández-Irigoyen
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  4. Fernando J. Corrales
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Corresponding authors

Correspondence to Enrique Santamaría or Fernando J. Corrales .

Editor information

Editors and Affiliations

  1. Warren Alpert Medical School, Proteomics Core Facility, Brown University, Hoppin Street 1, Providence, 02903, Rhode Island, USA

    Djuro Josic

  2. Div. Hematology & Oncology, Rhode Island Hospital, Eddy St. 593, Providence, 02903, Rhode Island, USA

    Douglas C. Hixson

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Santamaría, E., Sánchez-Quiles, V., Fernández-Irigoyen, J., Corrales, F.J. (2012). A Combination of Affinity Chromatography, 2D DIGE, and Mass Spectrometry to Analyze the Phosphoproteome of Liver Progenitor Cells. In: Josic, D., Hixson, D. (eds) Liver Proteomics. Methods in Molecular Biology, vol 909. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-959-4_12

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  • DOI: https://doi.org/10.1007/978-1-61779-959-4_12

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-958-7

  • Online ISBN: 978-1-61779-959-4

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