Site-specific peptide immobilization strategies for the rapid detection of kinase activity on microarrays.

The massive throughput offered by array-based technologies can only be realized with the development of equally powerful strategies that offer reproducible consistency. The competence of arrays and efficacy of screening come under scrutiny, with most existing immobilization schemes that do not site-specifically ligate peptides on the arrays. Thus, it is crucial in array-based experiments to orientate peptides in an ordered and uniform fashion.

Site-specific immobilization of biotinylated proteins for protein microarray analysis.

The postgenome era has led to a new frontier of proteomics that requires the development of protein microarray, which enables us to unravel the biological function of proteins in a massively parallel fashion. Several ways of immobilizing proteins onto surfaces have been reported, but many of these attachments are unspecific, resulting in the unfavorable orientation of the immobilized proteins. His6 tag has been used to site-specifically immobilize proteins onto nickel-coated slides, which presumably oriented proteins uniformly on the surface of the slide.

Intein-mediated biotinylation of proteins and its application in a protein microarray.

We report here the first example using an intein-mediated expression system to generate biotinylated proteins suitable for immobilization onto avidin-functionalized glass slides. With this novel array, proteins are site-specifically immobilized on the glass surface and are able to retain their native activity. The advantage of the avidin/biotin linkage over his-tag/Ni-NTA strategies for protein immobilization is highlighted by its ability to withstand a variety of chemical conditions, which makes this new protein array compatible with most biological assays.

Antibody-based fluorescence detection of kinase activity on a peptide array.

Peptide-based microarrays allow for high-throughput identification of protein kinase substrates. However, current methods of detecting kinase activity require the use of radioisotopes. We have developed a novel fluorescence-based approach for quantitative detection of peptide phosphorylation on chip using fluorescently-labeled anti-phosphoserine and anti-phosphotyrosine antibodies.

Developing site-specific immobilization strategies of peptides in a microarray.

In peptide-based microarrays, most existing methods do not allow for site-specific immobilization of peptides on the glass surface. We have developed two new approaches for site-specific immobilization of kinase substrates onto glass slides: (1) slides were functionalized with avidin for attachment of biotinylated peptides; and (2) slides were functionalized with thioester for attachment of N-terminally cysteine-containing peptides via a native chemical ligation reaction.