Abstract
Methods for analyzing the phosphorylation status of proteins are essential to investigate in detail key cellular processes, including signal transduction and cell metabolism. The transience of this post-translational modification and the generally low abundance of phosphoproteins require specific enrichment and/or detection steps prior to analysis. Here, we describe three gel-based approaches for the analysis of differentially expressed phosphoproteins. These approaches comprise (1) the sequential fluorescence staining of two-dimensional (2-D) gels using Pro-Q® Diamond and SYPRO® Ruby dyes to visualize and quantify phosphoproteins in total cellular lysates as well as (2) affinity enrichment of phosphoproteins in conjunction with sequential fluorescence staining of the 2-D gels and (3) affinity enrichment of proteins prior to pre-electrophoretic fluorescence labeling and 2-D gel electrophoresis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Machida M, Kosako H, Shirakabe K et al (2007) Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis. FEBS J 274:1576–1587
Raggiaschi R, Lorenzetto C, Diodato E et al (2006) Detection of phosphorylation patterns in rat cortical neurons by combining phosphatase treatment and DIGE technology. Proteomics 6:748–756
Artemenko KA, Bergstrom Lind S, Elfineh L et al (2011) Optimization of immunoaffinity enrichment and detection: toward a comprehensive characterization of the phosphotyrosine proteome of K562 cells by liquid chromatography-mass spectrometry. Analyst 136:1971–1978
Morandell S, Stasyk T, Grosstessner-Hain K et al (2006) Phosphoproteomics strategies for the functional analysis of signal transduction. Proteomics 6:4047–4056
Kotlo K, Johnson KR, Grillon JM et al (2012) Phosphoprotein abundance changes in hypertensive cardiac remodeling. J Proteomics 77:1–13
Eustace AJ, Dowling P, Henry M et al (2011) 2D-DIGE analysis of phospho-enriched fractions from dasatinib-treated melanoma cell lines. J Proteomics 74:490–501
Delom F, Chevet E (2006) Phosphoprotein analysis: from proteins to proteomes. Proteome Sci 4:15
Schulenberg B, Aggeler R, Beechem JM et al (2003) Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye. J Biol Chem 278:27251–27255
Steinberg TH, Agnew BJ, Gee KR et al (2003) Global quantitative phosphoprotein analysis using multiplexed proteomics technology. Proteomics 3:1128–1144
Bergstrom Lind S, Artemenko KA, Elfineh L et al (2011) Toward a comprehensive characterization of the phosphotyrosine proteome. Cell Signal 23:1387–1395
Andersson L, Porath J (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal Biochem 154:250–254
Talvas J, Obled A, Sayd T et al (2008) Phospho-proteomic approach to identify new targets of leucine deprivation in muscle cells. Anal Biochem 381:148–150
Chen A, McEwen ML, Sun S et al (2010) Proteomic and phosphoproteomic analyses of the soluble fraction following acute spinal cord contusion in rats. J Neurotrauma 27:263–274
Stasyk T, Morandell S, Bakry R et al (2005) Quantitative detection of phosphoproteins by combination of two-dimensional difference gel electrophoresis and phosphospecific fluorescent staining. Electrophoresis 26:2850–2854
Tang W, Deng Z, Oses-Prieto JA et al (2008) Proteomics studies of brassinosteroid signal transduction using prefractionation and two-dimensional DIGE. Mol Cell Proteomics 7:728–738
Steinberger B, Besenfelder U, Brem G et al (2013) Comparison of gel-based phosphoproteomic approaches to analyse scarce oviductal epithelial cell samples. Proteomics 13:12–16
Blum H, Beier H, Gross HJ (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93–99
Acknowledgements
This work was funded by Life Science Calls, Niederösterreichische Forschungs-und BildungsgmbH (NFB).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Steinberger, B., Mayrhofer, C. (2015). Principles and Examples of Gel-Based Approaches for Phosphoprotein Analysis. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 1295. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2550-6_23
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2550-6_23
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2549-0
Online ISBN: 978-1-4939-2550-6
eBook Packages: Springer Protocols