P141-T A New Array Format for Protein Kinase Substrate Determination

Peptide arrays are useful tools to characterize antibodies, enzyme substrates or sequence specificities of interaction partners with given peptide sequences (e.g., SH2, SH3, MH2 and other domains). Here we present a new method¹ that allows production of hundreds of identical peptide arrays from a single synthesis run on modified, individual cellulose-disks. The disks are dissolved in the acid cleavage-mixture after synthesis and the resulting solutions of peptide-cellulose-conjugates are then spotted onto multiple slides by conventional spotting techniques. As application example we show results obtained with arrays of kinase substrate libraries and various consensus sequences of known kinase targets. These arrays can be used with different detection methods to profile known and unknown kinases for their substrate specificity.The new arrays are derived from the the well known SPOT method² but offer several major improvements: A smaller volume of sample (only 100 μL) is needed for incubation, and a high number of identical copies of the arrays enables large scale, parallel screening experiments. The cost of an individual array is considerably lower than that of a SPOT membrane. Unlike DNA hybridization, protein-protein interactions frequently suffer from low binding affinities. The new cellulose substrate with peptides linked to it generates a three dimensional scaffold on the array support with a peptide loading exceeding that of a monolayer by a factor of 100. The high peptide density of the spots should be advantageous to identify protein-interaction sites, even if their binding constants are low.

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