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

ABSTRACT

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. Two new approaches were developed for the directed immobilization of peptides on a microarray, by exploiting measures involving native chemical ligation reactions as well as biotin-streptavidin interactions. This makes it possible to stably immobilize peptides in a consistent manner and in a predetermined orientation on the microarray. The first scheme employs glass slides that are functionalized with avidin for attachment of terminally biotinylated peptides. The second uses slides containing thioester moieties to ligate N-terminal cysteine containing peptides. The authors successfully immobilized peptides on chip using these strategies, and, in extending their method to the study of kinase activity on microarrays, they also developed a novel detection scheme that abrogates the dependence on traditional radioactivity-based kinase screening assays. This method employs fluorescently labeled antiphosphoserine and antiphosphotyrosine antibodies in assessing and monitoring kinase activity on arrays. The above methodologies provide for a fast and sensitive approach with which to conveniently assess kinase activity using peptide microarrays.