Peptide microarrays for the determination of protease substrate specificity.
A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.
Salisbury
Cleo M
CM
Center for New Directions in Organic Synthesis, Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Maly
Dustin J
DJ
Ellman
Jonathan A
JA
D000596:Amino Acids / Q000737:chemistry; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D005449:Fluorenes / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010091:Oximes / Q000737:chemistry / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D003374:Coumarins; D005449:Fluorenes; D005456:Fluorescent Dyes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D009842:Oligopeptides; D010091:Oximes; D012697:Serine Endopeptidases; D013917:Thrombin