Research Methods

Proteomics I: An Introduction to Two-Dimensional Gel Electrophoresis

By Michael B. Fessler, MD

National Jewish Medical and Research Center

University of Colorado Health Sciences Center

Proteomics is a methodology that seeks to identify and characterize the protein complement of the genome, ranging from the descriptive cataloguing of cell/tissue protein expression, to physical, functional and interactive mapping of proteins within subcellular compartments. While there has been a recent profusion of new, sophisticated technologies in the field of proteomics, the technique which is perhaps the oldest - two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by peptide mass fingerprinting (PMF) - arguably remains unrivaled for its combination of resolving power, quantitative information, indication of post-translational modification, accessibility and, lastly, its relatively high-throughput efficiency.

The steps of the 2D-PAGE/PMF method to be discussed in this section are:

Protein separation

• 2D-PAGE
• Protein detection (staining)
• Gel image analysis to match and quantify identical protein spots among gels

Protein Separation
Overview of 2D-PAGE
2D-PAGE involves electrophoretic separation by isoelectric focusing (IEF), followed by orthogonal separation by molecular weight using SDS-polyacrylamide gel electrophoresis. IEF applies an electric potential across a pH gradient, exploiting the fact that proteins have varying isoelectric points (pI) - that pH at which the net charge of their amino side chains and amino and carboxyl termini is zero. As proteins are net-positive when pH<pI and net-negative when pH>pI. Several IEF apparati are commercially available (e.g., Amersham Pharmacia Biotech IPGphor, Biorad PROTEAN IEF Cell), as are pre-cast first-dimension 'IPG' polyacrylamide gel strips (e.g., Amersham Immobiline DryStrips).

Sample Preparation for 2D-PAGE
The greatest restrictions upon sample composition in 2D-PAGE are encountered in the first dimension. Because IEF relies upon charge, sample salt content must generally be <10 mM, and the IEF sample buffer must be non-ionic or zwitterionic. Hence, SDS cannot be used except in very dilute concentrations (i.e., <0.25%). Solubilization is consequently a particular problem for hydrophobic proteins, which, as a rule, are poorly represented in 2D-PAGE (1). Use of a non-ionic chaotrope such as urea greatly improves disruption of hydrogen bonds and hydrophobic interactions. The addition of thiourea, a stronger denaturant than urea itself on a molar basis, in a urea/thiourea combination (e.g., 8M urea and 2M thiourea) has been shown both to improve the solubility of membrane and nuclear proteins, and to improve protein transfer to the second dimension gel (2).

An example of an effective IEF sample buffer follows:

• 8M urea
• 2M thiourea
• 4% CHAPS
• 50 mM DTT
• 0.2% ampholyte cocktail
• Bromphenol blue

Starting with the biologic sample of interest, any of several classical cell/tissue disruption methods can be used - detergent lysis, mechanical homogenization, etc. - in the presence of protease/phosphatase inhibitor cocktails. Protein precipitation can subsequently be performed (TCA, acetone, etc.), and the pellet resolubilized in IEF sample buffer. Alternatively, the sample can be desalted in isotonic sucrose, lysed/denatured directly with IEF sample buffer to which 25 mM spermine has been added, the nucleic acids pelleted by centrifugation, and spermine in the supernatant diluted to <6 mM so that its charge does not interfere with IEF (3). In the case of body fluids with low nucleic acid content (e.g., BALF), desalting can be performed by dialysis or spin buffer exchange. In the end, the prepared sample must be solubilized in IEF buffer with a volume limitation of ~350 ul per 18 cm long IPG strip (the rehydrated IPG strip volume), and then used to rehydrate IPG strips (>10 hours to overnight). Sample concentrations should not exceed 10 mg/ml, as higher concentrations may lead to protein losses as well as poor resolution. As a benchmark, depending on the sample, ~1-2.5 mg of protein loaded on an 18 cm IPG gel (i.e., ~2.9-7.1 mg/ml in 350 ul IEF buffer) generally achieves acceptable results.

Running the Gel

Specific IEF protocols are provided with commercially available IEF apparati, and will not be discussed here. A standardized volt-hours goal (typically between 25 and 100 Kvolt-hours for 18 cm IPG strips) should be used for like samples. Focusing time depends on gel length, pH gradient, ionic content, and protein load, and must be optimized empirically for each sample. Temperature should be strictly controlled (i.e., 20°C) as pI is temperature-dependent. Hydrated filter paper wicks at the electrodes (optional) can function as ion traps, sequestering ionic components away from the IPG gel strip, and should especially be considered for narrow pI range "zoom" gels (discussed below). Covering the IPG strip with mineral oil prevents desiccation and associated urea crystallization.

After completion of IEF, the IPG strip is prepared for second-dimension SDS-PAGE by immersion in SDS equilibration buffer. Generally, two 10-15 minute washes are performed, the first in a DTT-containing buffer, the second in an iodoacetamide-containing buffer. This loads the sample with SDS, and reduces/alkylates sulfhydryl moieties. The IPG strip is then positioned on a vertical or horizontal SDS-PAGE apparatus, and electrophoresis carried out for 4-24 hours, depending on achievable voltage and cooling conditions. While IPG strips of varying lengths up to 24 cm are commercially available and provide increasing first-dimension resolving power, shorter strips (e.g., 7 cm) may be perfectly adequate for less complex samples, and carry the virtue of producing denser protein spots, improving enzyme effect in the subsequent in-gel digest.

Perhaps the greatest challenge for 2D-PAGE, and proteomics in general, is the range of protein expression encountered in biological samples, estimated to exceed 7 orders of magnitude. By contrast, silver and Coomassie stains have a linear dynamic range of < 2 orders of magnitude. While use of pI 3-10, 18-24-cm wide gels may be optimal for initial screening of complex samples (e.g., lysates), simplification of the proteome (e.g., immunoprecipitation) enriches for less abundant proteins. Alternatively, commercially available narrow pI range IPG strips (e.g., pI 5.0-6.0 over 18 cm - "zoom gels") enhance spot resolution and permit protein loads up to 10 mg.

Figure 1. pI 3-10 two-dimensional polyacrylamide gel of
lipopolysaccharide-stimulated human neutrophils (left panel),
with corresponding pI 5.0-6.0 "zoom gel" (right panel)

Protein Detection: Which Stain to Use?
A comparison of the most commonly encountered detection methods used in 2D-PAGE follows:

While the limited sensitivity of Coomassie Brilliant Blue G-250 makes it a poor choice for protein detection, sensitivity is greatly enhanced if it is allowed to form colloidal microprecipitates (4). While the sensitivity of silver staining makes it a good choice for analytical gels (i.e., sample load <50-100 µg), in which the goal is simple comparison of protein expression patterns and not protein identification, sensitivity is not as good in protocols which omit aldehyde fixation in order to permit subsequent mass spectroscopic protein identification. Furthermore, concerns have been raised as to the reproducibility of silver staining, and its extremely narrow linear dynamic range dramatically limits quantitation. By contrast, fluorescent stains (e.g., SYPRO) have sensitivity rivaling that of silver, and far greater linear dynamic range, making them both the best-performing and most expensive stains currently available.

Image Analysis of 2D-Gels
If qualitative or quantitative comparison of protein expression patterns on different 2D gels is the goal of a proteomics experiment, scanning of gels followed by software-driven image analysis is recommended. Image analysis packages perform spot detection, enumeration, cataloguing, quantitation, matching between different gels, and basic statistical functions. Multiple packages are commercially available:

A recent alternate approach to the challenge of inter-gel spot matching is that of differential gel electrophoresis (DIGEâ„¢), marketed by Amersham Pharmacia Biotech . This method uses different cyanine dyes (i.e., Cy2, Cy3 and Cy5) characterized by differing peak excitation wavelengths, to fluorescently conjugate proteins from different samples. The two samples are subsequently mixed and run together on a single 2D gel. Identical proteins from the two samples co-migrate, and imaging of the gels using different excitation/emission filters can be used to differentially quantitate protein expression, spot by spot, in the original two samples.

Suggested Readings

• Gorg A, et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000; 21:1037-1053.
• Mann M, et al. Analysis of Proteins and Proteomes By Mass Spectrometry. Annu Rev Biochem 2001; 70:437-473.
• Rabilloud, S. Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods, (New York) 2000.

Useful Websites

• http://ca.expasy.org/tools/ (compilation of numerous useful links)
• www.lecb.ncifcrf.gov/flicker/
• http://ca.expasy.org/ch2d
• http://proteomics.cancer.dk/
• www.ionsource.com/

REFERENCES

1. Adessi C., et al. Two-dimensional electrophoresis of membrane proteins: a current challenge for immobilized pH gradients. Electrophoresis 1997; 18(1):127-135.
2. Rabilloud T, et al. Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 1997; 18(3-4): 307-316.
3. Rabilloud T, Valette C, Lawrence JJ, Sample application by in-gel rehydration improves the resolution of two- dimensional electrophoresis with immobilized pH gradients in the first dimension. Electrophoresis 1994; 15(12):1552-1558.
4. Neuhoff V, et al. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988: 9(6): 255-262.
5. Rosenfeld J, et al. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 1992: 203(1):173-179.