The aim of this review is to highlight the latest developments in the preparation, analysis and biotechnological applications of protein microarrays. Just before MacBeath and Schreiber reported for the first time the use of protein microarrays in 2000 (10
), the concept of using protein microarray technology was simply regarded as a dream. A decade later, the number of publications on protein microarray technologies has increased dramatically. There are approximately 32,000 publications indexed in PubMed (http://www.ncbi.nlm.nih.gov/pubmed
) under the keyword protein microarrays
. We have seen numerous examples that show protein microarrays are a very valuable tool for the study of whole proteomes (11
), protein identification and profiling for early diagnosis of diseases such as cancer (16
) or viral infections (158
) and for drug identification and validation (159
Despite the large number of successful examples in the use of protein microarrays in biomedical and biotechnological applications during the last 10 years, there are still, however, some challenges that need to be tackled. For example, most of the methods commonly employed for the immobilization of proteins onto solid supports rely on non-site-specific immobilization techniques (10
). The use of these methods usually results in the proteins being displayed in random orientations on the surface, which may compromise the biological activity of the immobilized proteins and/or provide false results (162
). This issue has been addressed over the last few years by the development of novel site-specific immobilization approaches which involve the use of chemoselective ligation reactions (52
), active site-directed capture ligands (112
) and protein splicing (68
), among others.
The expression and purification of thousands of proteins without compromising their structural and biological activity is also a challenging task. The use of cell-free expression systems in combination with nucleic acid arrays, which are more readily available and easier to prepare, has been shown to give good results to produce in situ
protein arrays from DNA (67
) and RNA arrays (130
). The combination of these approaches with site-specific and traceless methods of protein immobilization such as protein trans-splicing (68
) shows great promise.
The introduction of label-free detection methods, such as surface plasmon resonance and mass spectrometry, also shows great promise to simplify the use of protein microarray analysis, since labeling of the interacting partners will no longer be required.
The standardization of protein microarray production is another issue that needs to be improved. At this time, most of the methods used by the scientific community for preparing and analyzing protein microarrays are not completely standardized. The adoption of stringent standards by the scientific community for the production and analysis of these valuable reagents should, in principle, allow the generation of data that could be compared and exchanged across different studies and different research groups.
None of these challenges is impossible to achieve; in fact, as we have seen in this review, much more progress has been made over the last decade to address them. At this point, we strongly believe that the protein microarray technology is on the brink of becoming a standard technique in research in the same way as DNA microarray technology is used today.