Review
Strategies for revealing lower abundance proteins in two-dimensional protein maps

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Abstract

One of the most challenging contemporary research endeavors is the mapping of proteins and establishing their linkages to normal and pathological conditions. The availability of current proteomics technologies has greatly facilitated the separation and identification of proteins in a complex protein mixture by standard two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry. Due to the huge differences in the distribution of proteins in complex proteomes of humans, the detection and identification of proteins expressed in low copy number is a major challenge. The low abundance of important physiologically relevant proteins has rendered their analyses almost impossible without some means of prior purification and enrichment from tissue lysates or biological fluids. It is the current limits of detection of the methods that are used that prevents the detection of these proteins not the proteins themselves. More importantly, considering the frequency at which post-translational modifications of proteins occur, the separation of protein isoforms is essential to understand biological changes, and two-dimensional gel electrophoresis remains the only technique that can offer sufficient resolution to address this issue at a functional level. Cellular fractionation techniques followed by specific affinity probes for tracking target proteins have been developed to deplete the proteome of high abundance proteins in order to increase the sample loading for achieving greater sensitivity for proteins present in low abundance. Those applications can entail the removal of one protein or a class of proteins that interferes with the resolution of proteins in a 2-DE map. Moreover, the use of better solubilizing detergents in combination with an overlapping narrow immobilized pH gradients, results in higher resolution by stretching the protein pattern in the first dimension. In this review we will discuss strategies to remove high abundance proteins that can result in the visualization and detection of low abundance proteins in biological samples. The potential use of these strategies, as a means of developing diagnostic tools for early screening of diseases and identification of drug targets for therapeutic intervention, will also be discussed.

Introduction

The study of human proteomes, their diversity and their relationship to genome has raised great interest. The Human Genome project provides evidence for approximately 30,000–40,000 genes in the human proteome [1], a range that is not much in excess of the gene numbers in the fruit fly Drosophila melanogaster. The seemingly small number of genes in the human genome compared to other less complex organisms has evoked scientific debate about the extent of complexity and diversification of the human genome and its differences with other organisms such as bacteria, yeast, flies, other mammals, etc. It is apparent that the paradigm of ‘one gene encoding a single protein’ is no longer applicable because of differential RNA processing such as alternative RNA splicing, trans-slicing RNA events, overlapping transcription events, etc. [2]. The end result of these processes includes post-translational protein modifications resulting in multiple protein products for a single encoded gene. Hence, for every eukaryotic genome studied to date, the actual number of distinct proteins formed in the differing proteomes during the development far exceeds the number of transcription units that encoded them. Additionally, protein degradation and turnover can significantly influence the intracellular concentration of active protein molecules. It has been estimated, that the average number of proteins per gene is one or two in bacteria, three in yeast and three to more than six in human [3]. Hence, the extent of diversity and complexity resulting from post-translational modification and degradation is tremendous and can only be understood by qualitative and quantitative analysis of gene expression at the level of functional protein. Therefore, a direct measurement of protein expression in different proteomes is essential to analyze and understand biological processes during development, in disease and in normal conditions. The interface between protein biochemistry and molecular biology for the global analysis of gene expression and subsequent post-translational modification of product per se is termed ‘proteomics’ [4]. Proteomics is a technology-based science that studies in a high-throughput mode the expression of different proteins in a proteome, modifications and interaction of proteins that occur due to changes in the proteome during the developmental process, disease-state or exposure to an external stimuli. The core element of proteomics analysis is to combine the separation of proteins in a two-dimensional map together with mass spectrometry (MS) or tandem MS (MS–MS) protein identification. The major advantage of this technique is that it enables the simultaneous separation, visualization and identification of hundreds of unknown proteins at different modification states. No other method is able to achieve this at the present time. The technology has successfully been applied to gain understanding of the protein profile of simple organisms such as M. genitalium [5], E. coli [6], yeast [6], etc. Characterizing the proteome of a complex organism such as humans however, challenges the limitations of currently available technologies.

One of the challenges in identifying proteins in a complex proteome such as human is the presence of proteins in wide dynamic concentration ranges from very high levels for albumin to low for a specific hormone or a regulatory protein. The differences in the concentration of such proteins may be over thousands to a million times. Hence, a small sample volume (about 10–100 μl) usually applied for proteomics analysis, results in the acquisition of data dominated by high abundance proteins leaving a large percentage of the expressed proteins with insufficient quantities undetected. This large percentage of protein expressed in lower concentration constitutes of low abundance protein and includes proteins required for important regulatory processes or may represent proteins with potential biological marker possibility or may be a likely target for drug intervention. To detect these low abundance proteins of greater biological importance there is an urgent need to develop methods that will remove and deplete the relevant proteome of high abundance proteins, resulting in the analysis of 3–5-fold more proteins present in lower concentration. Strategies to effectively remove high abundance proteins to identify proteins of low abundance therefore have gained much interest.

Section snippets

Strategies for the enrichment and identification of low abundance proteins

As discussed above, proteomics is an area of research that seeks to define the function and relative expression profiles of proteins encoded by a given genome at a given time in a given cellular location. The technology separates, identifies, and characterizes the proteins expressed, retained, secreted or released by a cell or tissue in order to establish their function(s) and potential relationship during developmental phase and or onset or progression of diseases, as well as relapse and/or

Sample preparation and selection

An optimized sample preparation procedure for the visualization of low abundance protein in tissue samples includes preparation and purification of the cell type of interest. This will result in optimal cell lysis and solubilization of protein to gain optimal yield of low abundance protein. Sample selection is of paramount importance, for example use of fresh samples for 2-DE analysis was reported to be better than working with frozen samples [9]. Prolonged storage of samples may result in the

Strategies to enrich low abundance protein in biological fluids

The introduction of new technologies for the detection of disease specific biomarkers in the biological fluids of patients will have an important impact on the health sector. This need is particularly urgent in cancer and other diseases where early diagnosis dramatically improves patient outcome [44].

Blood transports essential nutrients to the cells and carries away metabolic waste products and other substances. Hence, blood proteins are useful diagnostic tools and alteration of the expression

Advances and limitations of 2-DE based technology

A number of methodological improvements have been made to 2-DE technology since its introduction by O’Farrell and Klose in 1975. The development of immobilized pH gradients of different ranges has made the technique more reproducible and allowed comparison of results between inter-laboratory groups. This improvement has also resulted in increasing the loading capacity of a sample making the technique more sensitive for the detection and identification of low abundance protein. The resolution of

Future perspectives and conclusions

The focus in biomedical research is to identify early stage markers for the diagnosis and better therapeutic treatment of diseases or to determine the molecular defect that underlines a specific disease or to identify potential targets for drug treatment. Proteomics is used as a technology to solve the underlying basis of all these problems. The basis of the technology is not only to list differentially expressed proteins in a proteome, but the technology acts as a circuit where the

Acknowledgements

The authors wish to acknowledge the technical help of Ms. Karen Oliva and Ms. Gillian Barker. The proteomics study is supported by the Jack Brockhoff Foundation, the Cancer Council of Victoria, the Rotary Club of Williamstown, Ovcare, Jigsaw Women's Fashion Company, B.O.O.T.S. ALL Inc. (Breasts Ovaries and other things sacred) and BHP Billiton Community Trust Australia.

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