Current Proteomics - Volume 1, Issue 4, 2004

Separation of proteins from biological samples by two-dimensional polyacrylamide gel electrophoresis (2D -PAGE) followed by their identification by mass spectrometry is a widely used technique in proteomics. Two dimensional difference gel electrophoresis (2D-DIGE) is a relatively new technique in 2D-PAGE for multiplex quantitative analysis of the component proteins of related but different protein samples. In this technique, prior to electrophoresis, the protein samples are labeled with different fluorescent dyes that have distinct spectral characteristics. The labeled samples are subsequently combined and then subjected to gel electrophoresis. Fluorescent dyes are available for this purpose that are matched for charge and mass. This enables co-separation, co-detection and simultaneous quantitative estimation of the components of different protein samples on the same gel. Thus, DIGE is useful for multiplex analysis. Commercially available CyDye DIGE Fluor minimal dyes have been widely used in 2D-DIGE in order to study simultaneous expression of protein molecules from related but different samples. On the other hand, CyDye DIGE Fluor saturation dyes are more recently available dyes used for such purpose and are particularly useful for studying biological samples, which are available in limited quantity. The chemistry for labeling the protein samples with CyDye DIGE Fluor minimal dyes is different from that for labeling the protein samples with CyDye DIGE Fluor saturation dyes. The present article will discuss the advantages and disadvantages of these dyes as well as the application of DIGE in the rapidly growing area of quantitative proteomics research.

The proteome is defined as the set of proteins that an organism can produce. By extrapolation, the “Proteome Phenotype” corresponds to the set of proteins whose synthesis is modified (under- or over-expressed) in a genetically altered cell. This approach has been successfully used to identify co-regulated genes in some Gram positive and Gram negative bacteria but can also be carried out with cells mutated in non regulatory genes in parallel to physiological phenotype studies. Over past ten years, such results have been obtained with several mutant strains of Enterococcus faecalis, a rogue commensal-turned-pathogen and Gram positive bacteria. Systematic 2-D gel electrophoresis carried out with cells affected in genes encoding stress proteins or transcriptional regulators such as CcpA, HypR, or TCSs (Two Component Systems) allowed to characterize some members of these regulons and to identify pleiotrophic effects of some mutations. Moreover, comparison of gels obtained with proteins extracted from cells harvested after exposure to different stress conditions revealed “Stress Proteome Signatures” of E. faecalis. The availability of the E. faecalis proteome, updated in this review, is the key database to utilize these results for biomedical applications. Indeed, 46 proteins are now characterized on a master gel which allowed identification of some effectors of the stress response. Because E. faecalis is a member of the gastrointestinal tract flora, such proteomics approach may help to understand how it is able to cope with different hostile environments and then become a pathogen responsible for infections in the bloodstream, urinary tract, or surgical wound infections.

Microarray technology plays an increasing role in proteomic research. We give an overview about recent developments in this technology focusing on molecular interaction studies using protein and antibody microarrays. We report about technical aspects in the development of protein microarrays and describe different surfaces and detection modes. Furthermore, we review the applications of protein microarrays in different molecular interaction studies including interactions of proteins with antibodies, proteins, DNA, small molecules and enzymes. Advantages and limitations of the microarray-based methods with other in vitro methods have been compared. We present the increasing applications of protein and antibody microarrays in basic research, diagnostics, drug discovery, and in vitro-risk assessment of nutrients.

In recent years, there has been a rapid and steady increase in the number of nucleic acid binding proteins (NABPs) and the list is continually increasing. However, a significant percentage of NABPs, the so-called hypothetical or predicted proteins has only been presumed based upon nucleic acid sequences and therefore remain to be shown to exist at the protein level. In general, neither the expression pattern analysis nor a protein hunting strategy to validate these proteins has been reported. In the present article, we summarize our studies on the application of a proteomics approach to document the identification and expression patterns of several of these proteins in a series of individual cell lines. The power of the two dimensional gel electrophoresis with subsequent in-gel digestion of protein spots followed by mass spectrometry based identification was used for the concomitant demonstration and verification of a series of NABPs in bronchial epithelial, amnion, mesothelial, lymphocyte, and kidney cell lines. Several heterogeneous nuclear ribonucleoproteins (e.g. A/B, R), Non-POU-containing octamer-binding protein, similar to paraspeckle protein, Alf-C1, similar to NS-1 associated protein 1, proteins with septin domains, GTP-binding proteins, components of the splicing, transcription and translation machinery as well as NABPs with still unknown function were unambiguously identified and these contribute to the many pathways and cascades of NABPs in a cell specific pattern.

In the post-genome era, real-time monitoring of proteins is of great importance. The application of molecular beacons (MBs) for real time protein detection demonstrates great advantages in the understanding of many important biological processes involving two key biomolecules: nucleic acid and protein. In general, MBs are single-stranded oligonucleotide probes that have been designed to report the presence of complementary nucleic acids by fluorescence detection. MBs have also been used for protein recognition and protein/DNA interaction studies. Real-time monitoring of enzymatic reactions, such as the cleavage of single-stranded DNA by single-strand specific nucleases, has been effectively studied by MBs. The excellent signal transduction properties of MBs can be combined with the specificity of aptamers for protein binding for the development of a new generation of molecular probes, molecular beacon aptamer (MBA), for molecular recognition of proteins and for specific protein binding studies. Using this principle, new fluorescent probes have been developed for real-time protein monitoring, such as the MBA for ultrasensitive real-time monitoring of the platelet derived growth factor. These probes are highly selective and sensitive with detection limits in the sub-nanomolar range. MBs have also found unique applications in studying ligation and phosphorylation in real time and with excellent specificity.

This review focuses on rapid, efficient, and sensitive characterization of the proteome by capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE) in conjunction with laser-induced fluorescence (LIF). A number of advanced CE-LIF and MCE-LIF techniques, including on-column concentration techniques, on-line chemical reaction and enzyme-assay techniques, integrated microdevices, and high-efficiency multidimensional separation techniques that have been developed within the past few years, are capable for the analysis of trace proteins in biological samples and/or in single cells. These powerful techniques also show great potential for drug screening and diagnosis. The basic principle of each technique and its advantages and shortcomings for proteomics studies are discussed in detail. We also highlight the importance of techniques that possess improved analytical sensitivity, sample throughput, and quantitation capabilities for identifying the complexities and dynamics of the proteome expressed by cells, tissues, or an organism.