Protein chip technology

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Abstract

Microarray technology has become a crucial tool for large-scale and high-throughput biology. It allows fast, easy and parallel detection of thousands of addressable elements in a single experiment. In the past few years, protein microarray technology has shown its great potential in basic research, diagnostics and drug discovery. It has been applied to analyse antibody–antigen, protein–protein, protein–nucleic-acid, protein–lipid and protein–small-molecule interactions, as well as enzyme–substrate interactions. Recent progress in the field of protein chips includes surface chemistry, capture molecule attachment, protein labeling and detection methods, high-throughput protein/antibody production, and applications to analyse entire proteomes.

Introduction

The past ten years have witnessed a fascinating growth in the field of large-scale and high-throughput biology, resulting in a new era of technology development and the collection and analysis of information. The challenges ahead are to elucidate the function of every encoded gene and protein in an organism and to understand the basic cellular events mediating complex processes and those causing diseases 1., 2., 3., 4.. Miniaturized and parallel assay systems, especially microarray-based analyses, are crucial to large-scale and high-throughput biological analysis, as they are a rapid and economic way to interpret gene function 3., 5., 6., as demonstrated by DNA microarray approaches 7., 8.. In a microarray format, capture molecules are immobilized in a very small area, and probed for various biochemical activities. High signal intensities and optimal signal-to-noise ratios can be achieved under ambient analyte conditions [3]. The microarray format has become the leading technology that enables fast, easy and parallel detection of thousands of addressable elements and side-by-side measurements.

Despite the success of DNA microarrays in gene expression profiling and mutation mapping, it is the activity of encoded proteins that directly manifest gene function. Thus, one would expect protein microarrays, in which proteins are prepared, arrayed and analysed at high spatial density, to be particularly powerful for analysing gene function, regulation and a variety of other applications. Proteins are more challenging to prepare for the microarray format than DNA, and protein functionality is often dependent on the state of proteins, such as post-translational modifications, partnership with other proteins, protein subcellular localization, and reversible covalent modifications (e.g. phosphorylation). Nonetheless, in recent years there have been considerable achievements in preparing microarrays containing over 100 proteins and even an entire proteome 1., 2., 9., 10., 11.. Alternative array formats have also been developed including tissue arrays [12], living cell arrays 13.•, 14.•, peptide arrays 1., 15., 16., 17., 18.••, antibody/antigen arrays 19.••, 20., protein arrays 21., 22., 23.••, 24.••, 25.••, carbohydrate arrays 26.••, 27.••, and small-molecule arrays [28••]. However, technological challenges in the field of protein microarrays still remain.

In this review, we discuss recent progress in the field of protein chips, including surface chemistry, capture molecule attachment, protein labeling and detection methods, high-throughput protein/antibody production, and applications to analyse protein families and entire proteomes.

Section snippets

Manufacture of protein chips

It is important that protein chips retain proteins in an active state at high densities, are compatible with most commercial arrayers and scanners, and can be printed in such a fashion that the proteins remain in a moisturized environment. Soft substrates such as polystryrene, poly(vinylidene fluoride) (PVDF), and nitrocellulose membranes, which have been used to attach proteins in traditional biochemical analyses (e.g. immunoblot and phage display), are often not compatible for protein

Probe detection methods

Fluorescence detection methods are generally the preferred detection method (Table 2) because they are simple, safe, extremely sensitive and can have very high resolution 1., 3.. They are also compatible with standard microarray scanners. Typically, a chip is either directly probed with a fluorescent molecule (e.g. protein or small molecule) or in two step by first using a tagged probe (e.g. biotin), which can then be detected in a second step using a fluorescently labeled affinity reagent

Two functional classes of protein microarrays

There are two general types of protein microarrays. Firstly, analytical microarrays in which antibodies, antibody mimics or other proteins are arrayed and used to measure the presence and concentrations of proteins in a complex mixtures. Secondly, functional protein microarrays, in which sets of proteins or even an entire proteome are prepared and arrayed for a wide range of biochemical activities.

Other analytical microarrays

In addition to antibody microarrays, other analytical microarrays have been developed. These include microarrays for profiling antibodies in a patient’s serum, essentially the reciprocal of that described above. Joos and colleagues [20] used 18 diagnostic markers for autoimmune diseases to form an autogen microarray and screened for antigen–antibody interactions. Hiller et al. [48] arrayed 94 purified allergen molecules, which included most common allergen sources, on glass slides to

Conclusion

Protein microarrays are poised to become one of the most powerful tools in the field of large-scale biology because of their enormous potential in basic research, diagnostics and drug discovery. High-density robotically spotted protein microarrays on glass have been validated to analyse an entire proteome and hold great promise for high-throughout discovery applications 19.••, 23.••, 25.••. Improvements in generating large sets of antibody reagents, recombinant proteins from a variety of host

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • of special interest

  • ••

    of outstanding interest

Acknowledgements

HZ is supported by a postdoctoral fellowship from the Damon Runyon-Walter Winchell Cancer Research Foundation. Research in the Snyder lab is supported by grants from the National Institutes of Health.

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