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Resolving acetylated and phosphorylated proteins by neutral urea Triton-polyacrylamide gel electrophoresis: NUT-PAGE
 
Christopher J. Buehl*1, Xiexiong Deng*2, Mengyu Liu*2, Stacy Hovde2, Xinjing Xu2, and Min-Hao Kuo1,2
1Cell and Molecular Biology Program, Michigan State University, East Lansing, MI
2Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI


701W 168th St. HHSC 1406, New York, NY 10032

*C.J.B., X.D., and M.L. contributed equally to this work.
BioTechniques, Vol. 57, No. 2, August 2014, pp. 72–80
Full Text (PDF)
Abstract

Protein acetylation and phosphorylation are key modifications that regulate both normal and pathological protein functions. The gel systems currently used for analyzing modified proteins require either expensive reagents or time-consuming second dimension electrophoresis. Here we present a neutral pH gel system that allows the analysis of acetylated and phosphorylated proteins. The neutral pH urea Triton-polyacrylamide gel electrophoresis (NUT-PAGE) system separates proteins based on their charge at pH 7.0 and generates discrete bands from each acetylated and/or phosphorylated species. In addition, the gel is composed of common and inexpensive laboratory reagents and requires only a single dimension of electrophoresis. We demonstrate the effectiveness of this system by analyzing the phosphorylated species of an acidic protein, α-synuclein, and both acetylated and phosphorylated species of a basic protein, histone H3. NUT-PAGE thus provides a cost-effective alternative for resolving acetylated and phosphorylated proteins, and potentially proteins with other post-translational modifications that alter net charge.

Protein lysine (Lys) acetylation and serine/threonine (Ser/Thr) phosphorylation are among the most pervasive post-translational modifications controlling the normal and even pathological functions of numerous proteins. It has been estimated that 30% of human proteins are phosphorylated at any given moment, and acetylation of histones and non-histones is increasingly found to play critical roles in a variety of cellular and nuclear functions, including metabolism, nutrient sensing, and gene regulation (1, 2).

A gel system that can effectively resolve acetylated and phosphorylated proteins is of tremendous value in biomedical research. While SDS-PAGE (sodium dodecyl sulfate-PAGE) (3) is an essential laboratory technique, in most cases it is unable to resolve protein isoforms resulting from modifications such as acetylation and phosphorylation. This is because acetylation and phosphorylation add only 42 and 80 daltons, respectively, to the protein. On the other hand, these two modifications reduce the net charge per modification site by one (acetylation) or one to two (phosphorylation) at physiological pH, rendering acetylated and phosphorylated proteins amenable to electrophoresis systems based on separation by protein charge. One such method is isoelectric focusing (IEF), in which proteins are introduced to a gel matrix with a stable pH gradient generated by ampholytes. Electric current pushes proteins to migrate until they reach the zone where the pH is equivalent to the isoelectric point (pI) of the protein (4). IEF can resolve proteins differing by a small charge variation. However, proteins with similar charges may differ significantly in their sizes. To further separate such proteins, IEF is paired with a second dimension, SDS-PAGE (5), increasing the cost and labor of the assay.

METHOD SUMMARY

Here we present a single-dimension neutral pH urea Triton-polyacrylamide gel electrophoresis (NUT-PAGE) system affording high-resolution separation of acetylated and phosphorylated proteins.

In addition to IEF, other charge-based gel systems are available as well. These systems typically use urea to denature proteins and various chemicals and buffers to maintain a set pH in the gel and the running buffer so that proteins are ionized and resolved by both size and charge. One of the most successful gel systems in this category is the Triton acetic acid urea gel (TAU). TAU-PAGE has been used widely to resolve acetylated histones (6-8). However, due to the acidity of the gel system, the phosphate groups of certain phosphorylated proteins might become protonated and less charged, rendering acid urea gels less effective in separating phosphoproteins. In principle, the charge differences caused by Lys acetylation and Ser/Thr phosphorylation are maintained at neutral pH. Thus, a gel system that runs at pH 7.0 should be a useful and versatile tool.

Protocols for urea-containing PAGE at or near neutral pH have been described previously (9, 10, 11). However, these methods are relatively underutilized, in particular in the study of post-translational modifications. In this paper, we present a neutral urea Triton-polyacrylamide gel electrophoresis system (NUT-PAGE). NUT-PAGE maintains neutral pH via the use of imidazole and MOPS (3-(N-morpholino)propanesulfonic acid), two inexpensive chemicals commonly found in biochemistry and molecular biology laboratories. For a proof of principle, we used α-synuclein, an acidic protein, and histone H3, a basic protein, to demonstrate the feasibility of NUT-PAGE in resolving both acetylated and phosphorylated proteins. This method provides a versatile and affordable alternative to IEF and 2-D systems.

Materials and methods

Cloning, protein expression, and purification

Human α-synuclein (AAS83394.1, GI:46242542) and budding yeast histone H3 (GI: 855700) were cloned into PIMAX system vectors developed in our laboratory (Sui et al., unpublished data), allowing the co-expression of a substrate with the cognate modifying enzyme and resulting in highly efficient modification of the substrate protein. α-synuclein was co-expressed with the human Aurora A kinase (NP_940835.1, GI:38327564), and histone H3 was co-expressed with either S. cerevisiae Gcn5 histone acetyltransferase (GI: 853167) or S. cerevisiae Ipl1 kinase (GI: 855892).

α-synuclein and histone H3 constructs were transformed into BL21–CodonPlus E. coli cells. All bacterial growth and induction steps were conducted in LB medium containing 100 µg/mL ampicillin. For induction, cells were seeded from an overnight culture at an OD600 of 0.1 and grown at 37°C until the OD600 reached 0.3. α-synuclein and histone H3 were induced by 0.5 mM and 0.25 mM IPTG, respectively, at 37°C for 2 h. Cells were then pelleted at 5000 × g at 4°C, resuspended in Buffer A (100 mM NaCl, 20 mM Tris-HCl, pH 7.4, 10% glycerol), and disrupted by sonication using a Misonix Sonicator 3000 (Farmingdale, NY) with 10 15-second bursts at 20% output.

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