Two-dimensional phos-tag zymograms for tracing phosphoproteins by activity in-gel staining.

Claudia-Nicole Meisrimler,Alexandra Schwendke,Sabine Lüthje

doi:10.3389/fpls.2015.00230

Abstract

Protein phosphorylation is one of the most common post-translational modifications regulating many cellular processes. The phos-tag technology was combined with two-dimensional zymograms, which consisted of non-reducing IEF PAGE or NEPHGE in the first dimension and high resolution clear native electrophoresis (hrCNE) in the second dimension. The combination of these electrophoresis methods was mild enough to accomplish in-gel activity staining for Fe(III)-reductases by NADH/Fe(III)-citrate/ferrozine, 3,3′-Diaminobenzidine/H2O2 or TMB/H2O2 in the second dimension. The phos-tag zymograms can be used to investigate phosphorylation-dependent changes in enzyme activity. Phos-tag zymograms can be combined with further downstream analysis like mass spectrometry. Non-reducing IEF will resolve proteins with a pI of 3–10, whereas non-reducing NEPHGE finds application for alkaline proteins with a pI higher than eight. Advantages and disadvantages of these new methods will be discussed in detail.

Highlights

Protein phosphorylation, one of the most common post-translational modifications, can alter enzyme activity and subcellular localization as well as target proteins for degradation and can effect changes in protein-protein interactions (Cousin et al, 2013; Gerbeth et al, 2013; Uhrig et al, 2013)
For non-equilibrium pH gel electrophoresis (NEPHGE) the pH gradient was directed in the opposite direction than for isoelectric focusing (IEF) (Figure 1)
NEPHGE is normally used for highly alkaline proteins that otherwise would be lost for any analysis by polyacrylamide gel electrophoresis (PAGE) and the following mass spectrometry (MS) identification

Summary

Introduction

One of the most common post-translational modifications, can alter enzyme activity and subcellular localization as well as target proteins for degradation and can effect changes in protein-protein interactions (Cousin et al, 2013; Gerbeth et al, 2013; Uhrig et al, 2013). Monitoring the phosphorylation status of proteins is, very important for the evaluation of diverse biological processes. Methods to quantify particular phosphorylation events include radioactive labeling, immunodetection of site-specific phosphorylations, phospho-specific site mapping in peptide mass fingerprinting, chemical labeling and in-gel phospho stainings (e.g., Pro-Q Diamond R , all blue and quercetin staining) (Ferrão et al, 2012; Wang et al, 2014). Complex protein samples are often separated by polyacrylamide gel electrophoresis (PAGE), before mass spectrometry (MS) analysis. After PAGE, immunodetection or phospho staining are the most commonly applied techniques to detect phosphorylated proteins

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Conclusion