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Applications of Protein Microarrays

Protein and Antibody Microarrays

Protein Chip Applications

 

Applications of Protein Chips
1) Proteomics
            Protein chip technologies will provide a powerful, high-throughput and versatile tool for the genome-scale analysis of gene function (see Figure 4).  Enzyme activity, protein–protein and protein–nucleic-acid interactions, and small-molecule drug interactions may all be analyzed directly on the protein level (77,78).  Arrays may be engineered to address protein identification, quantitation, and affinity studies.  A profiling array may quantitate levels of specific proteins on a global scale allowing for a comparison of normal and disease states.  An affinity array may analyze the interactions of peptides, proteins, oligonucleotides, sugars, lipids, or small molecules and chemicals with immobilized proteins such as receptors, enzymes, or antibodies (8). 
            Currently, the rate-limiting step is the production of large numbers of proteins. The ability to automate protein production and proteins fused to high-affinity tags will greatly expedite protein-chip development.  High-density chips containing large sets of proteins or even entire proteomes will allow the high-throughput analysis of biochemical activities, protein–protein interactions and post-translational modifications, such as phosphorylation, dephosphorylation, protein methylation, and ubiquitination.

            The ultimate goal of proteomics is to the study biochemical activities of every protein encoded by an organism or proteome.  A landmark study conducted prepared the first proteome chip by cloning ~94% (>5800 of 6200) of the yeast open reading frames in a yeast expression vector which expressed the proteins as N-terminal GST-His x6 double tagged fusions.  A high-throughput yeast protein purification method was developed to individually purify proteins. 80% of yeast proteins were full length and of sufficient quantity to be detectable by most assay types. The proteins were then purified using the GST tags and were then attached to Ni-NTA-coated glass slides using the HisX6 tags.  In addition to identifying known interactions, 33 novel binding proteins were detected.  150 novel lipid-binding proteins were also identified.  This study demonstrated that an entire proteome can be immobilized on a glass surface to directly screen for interactions with proteins and small molecules (26).
            The coupling of mass-spectrometry and protein chips will have wide applications in identifying players in protein–protein interactions, and also in drug discovery (80).  Proteins and small-molecule ligands bound to proteins immobilized on chip can be identified using matrix-assisted laser desorption/ionisation time of flight (MADLI-TOF) mass spectroscopy.  Microwell formats are particularly suited for this purpose. Thus, molecules and proteins that specifically bind to many different proteins can be identified and this information can be used to deduce molecular networks and pathways.
            One area that will require technological improvements is the analysis of membrane proteins.  A large amount of proteins are likely to be membrane-bound, since as many as one third of all yeast proteins are membrane proteins or secreted proteins (81). Due to the fact that many of these proteins are active when in membranes, it therefore may be necessary to purify or reconstitute them with associated lipids. However, this may not be so difficult. One group was able to immobilize biotinylated membranes that contain the G-protein-coupled receptor rhodopsin on a gold-coated glass surface, and establish a functional assay for that protein (82).  Similar procedures may make it possible to analyze membrane proteins in a chip format.
2) Diagnostics
Another area which will benefit from protein areas is diagnostics. Highly parallel analysis on arrays will allow determination of disease markers (e.g tumour markers) in extracts with only a minimum of biopsy (sample) material, creating new possibilities for monitoring disease (cancer) treatment and therapy (83).

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Next: Protein Microarrays: Future Directions and Conclusions

References for Protein and Antibody Microarrays

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Introduction and Background to Protein Chips and Antibody Chips.

Types of Antibody and Protein Chips

 

 

 

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