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.
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