Review
Methods of comparative proteomic profiling for disease diagnostics

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Abstract

The recent development of numerous technologies for proteome analysis holds the promise of new and more precise methods for disease diagnosis. In this review, we provide an overview of some of these technologies including two-dimensional gel electrophoresis (2DE), historically the workhorse of proteomic analysis, as well as some newer approaches such as liquid phase separations combined with mass spectrometry, and protein microarrays. It is evident that each method has its own strengths and weaknesses and no single method will be optimal in all applications. However, the continuing development of innovative strategies for protein separation and analysis is providing a wealth of new tools for multi-dimensional protein profiling that will advance our capabilities in disease diagnostics and our understanding of disease pathology.

Introduction

Technologies used for the parallel analysis of large numbers of proteins are advancing rapidly. The increasingly powerful tools for proteomic studies are providing new opportunities for the discovery of protein biomarkers that will be useful for diagnosing disease, monitoring disease progression or the efficacy of treatment, identifying new therapeutic targets, and understanding the underlying mechanisms of disease.

Nucleic acid based technologies have been widely used in studies of comparative gene expression profiling for biomarker discovery. However, it is essential that these studies also be carried out at the protein level. Proteins are the functional readout of genetic information and protein activity can be affected by many factors that are not reflected in the RNA transcript population (transcriptome). For instance, there can be a substantial discordance between mRNA abundance and protein expression levels [1], [2]. Further, over 200 different post-translational modifications [3] can regulate protein function by altering properties such as interactions with other biomolecules or sub-cellular localization. In developing tools for disease diagnostics, it is also important to consider that many of the biological fluids that are relatively accessible for analysis, such as serum, urine, and saliva, are rich in protein but very poor sources of nucleic acids for assay.

An ideal proteome screening methodology would combine high throughput capabilities with detection of as many protein products as possible in a sensitive, reproducible, and quantifiable manner. The wide-ranging biochemical heterogeneity of proteins makes it unlikely that any single separation and analysis method will be suitable for profiling the full proteome of any cell type, tissue, or biological fluid. In the following sections, we describe several of the tools that have been used or that are being developed for protein biomarker discovery and disease diagnostics, each with its own strengths as well as limitations. Several reviews of proteomic investigations in disease diagnosis have been published [4], [5], [6], [7], [8], [9]. Here, we will emphasize recent studies that are illustrative of proteomic approaches currently being used.

Section snippets

The basic technology

Two-dimensional gel electrophoresis (2DE), developed in the mid-1970s [10], [11], was the first method to allow the resolution and simultaneous display of hundreds of proteins. Recent improvements in the implementation of this basic technology, together with the explosion of protein sequence information resulting from genomic studies, and the development of techniques for peptide analysis by mass spectrometry, have fueled the emergence of proteomics as a powerful tool for comparative gene

Separation and analysis of proteins by liquid chromatography and mass spectrometry

There is a great deal of interest at the present time in developing gel-free systems for protein analysis because of their potential for multiplexing [51], [52]. An analogy may be made to DNA sequencing, notably as utilized in the genome project which received a considerable boost when the switch from gel-based approaches to a gel-free technology took place. Multi-modular combinations of HPLC, liquid-phase isoelectric focusing (IEF), and capillary electrophoresis (CE) provide various options to

Antibody and protein arrays

Antibody and protein arrays offer an attractive complement to separation and mass spectrometry methods for comparative proteomics research. Various technologies for probing binding interactions on arrays of immobilized antibodies, proteins or peptides are in development and use. Each technology has its own advantages, disadvantages, and optimal applications. These methods and their applications in comparative proteomics are reviewed here.

Acknowledgements

Work from the authors’ laboratories is funded, in part, by the National Cancer Institute Early Detection Research Network. L.F.S. receives additional support from NIH grant AI055988 and Nucleonics Inc. B.B.H. acknowledges support from the Cancer Research and Prevention Foundation, the Department of Defense (DAMD17-03-1-0044) and the Van Andel Research Institute.

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