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Comparative Proteomics and Difference Gel Electrophoresis
 
Jonathan Minden
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA
BioTechniques, Vol. 43, No. 6, December 2007, pp. 739–745
Full Text (PDF)
Abstract

The goal of comparative proteomics is to analyze proteome changes in response to development, disease, or environment. This is a two-step process in which proteins within cellular extracts are first fractionated to reduce sample complexity, and then the proteins are identified by mass spectrometry. Two-dimensional electrophoresis (2DE) is the long-time standard for protein separation, but it has suffered from poor reproducibility and limited sensitivity. Difference gel electrophoresis (DIGE), in which two protein samples are separately labeled with different fluorescent dyes and then co-electrophoresed on the same 2DE gel, was developed to overcome the reproducibility and sensitivity limitations. In this essay, I discuss the principles of comparative proteomics and the development of DIGE.

Introduction

What makes a stem cell distinct from its differentiated progeny? What makes a cancer cell behave differently from its normal neighbors? How do cells respond when exposed to different agents, such as drugs, viruses, or other cells? Whether these cellular changes are due to developmental, genetic, or environmental effects, they are ultimately mediated by changes in protein expression or modification. Mammalian cells contain thousands of different protein species, only a small number of which are changed under such circumstances. The primary goal of comparative, or differential, proteomics is to discover the protein changes that underlie these cellular changes.

Compared to genomics or transcript profiling, proteomics represents a much greater technological challenge, because the chemical complexity of the proteome is vastly greater than that of nucleic acids, and unlike nucleic acids, proteins cannot be amplified, which means that methods must be developed to handle minute amounts of protein. To appreciate the scale of chemical complexity of the proteome, let's consider the chemical space that cellular proteins occupy. For the purposes of this discussion, this protein chemical space can be defined by three axes: protein mass, protein isoelectric point (pl), and protein abundance. (Figure 1)A shows 1000 hypothetical proteins populating this chemical space, where protein mass ranges from 10,000 to 1,000,000 Da, pl varies from 3 to 10, and abundance spans 1000 to 10,000,000 copies/cell. To avoid overcrowding, this plot underestimates the number of protein species by at least an order of magnitude and does not take alternative splicing and posttranslational modification into account. In reality, this plot would have much more densely distributed spots.

Figure 1.


Simulation of protein chemical space. (A) Shows a simulation of 1000 proteins (black spots) distributed within a chemical space defined by protein isoelectric point (pl), molecular weight, and abundance. (B) Shows the addition of 50,000 tryptic peptide fragments (red spots) that result from the digestion of the protein spots. The spot size of the tryptic peptides are reduced relative to the protein spot so as not to overwhelm the image.

The challenge of comparative proteomics is to sift through this protein space and identify the few proteins that differ between the samples being compared. Proteome analysis has two essential components: protein fractionation and protein identification. Protein fractionation is necessary to reduce the protein complexity, so that the genes that encode the proteins of interest can be identified, and is typically done by column chromatography or gel electrophoresis. Protein identification is accomplished by mass spectrometry (MS). Although tremendous advances in MS technology have greatly improved our ability to identify protein fragments rapidly (1), fractionation is still necessary to reduce the complexity of the proteins, or their proteolytic fragments, prior to MS analysis.

Gel electrophoresis and column chromatography are the most common methods for protein fractionation. It is important to recognize that no fractionation scheme is capable of perfectly resolving all of the protein species found in cells or tissue—the chemical complexity is too great relative to the resolving power of these physical separation methods. Two or more orthogonal separation schemes are typically used to increase resolution. Two-dimensional electrophoresis (2DE) relies on separating proteins based on pl and mass. Many technological improvements have made 2DE relatively inexpensive and accessible to most biomedical research labs. Column chromatography offers a wide range of matrices for protein separation. Still, 2DE and multidimensional chromatography are not sufficient to resolve all cellular proteins. Column chromatography offers the advantage of automation and direct feed into MS. However, directly feeding column fractions into the MS limits the duty time allotted to analyze each fraction. Each fraction, therefore, represents a moving target. 2DE allows separated protein samples to be maintained within the gel, thus allowing more time to analyze each position within this separation space. An added advantage of 2DE is that several samples can be run simultaneously in a single apparatus, while column chromatography is limited to a single sample per apparatus. Thus, the sample throughput is much higher for 2DE gels than column-based separation methods.

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