Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays.
A novel microarray-based proteolytic profiling assay enabled the rapid determination of protease substrate specificities with minimal sample and enzyme usage. A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized and microarrayed, along with fluorescent calibration standards, in glycerol nanodroplets on microscope slides. The arrays were then activated by deposition of an aerosolized enzyme solution, followed by incubation and fluorometric scanning. The specificities of human blood serine proteases (human thrombin, factor Xa, plasmin, and urokinase plasminogen activator) were examined. The arrays provided complete maps of protease specificity for all of the substrates tested and allowed for detection of cooperative interactions between substrate subsites. The arrays were further utilized to explore the conservation of thrombin specificity across species by comparing the proteolytic fingerprints of human, bovine, and salmon thrombin. These enzymes share nearly identical specificity profiles despite approximately 390 million years of divergent evolution. Fluorogenic substrate microarrays provide a rapid way to determine protease substrate specificity information that can be used for the design of selective inhibitors and substrates, the study of evolutionary divergence, and potentially, for diagnostic applications.
Gosalia
Dhaval N
DN
Department of Bioengineering, Institute for Medicine and Engineering, University of Pennsylvania, PA, USA.
Salisbury
Cleo M
CM
Maly
Dustin J
DJ
Ellman
Jonathan A
JA
Diamond
Scott L
SL
D020816:Amino Acid Motifs; D000818:Animals; D002417:Cattle; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015394:Molecular Structure; D040081:Protein Array Analysis / Q000379:methods*; D012474:Salmon; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D003374:Coumarins; D005456:Fluorescent Dyes; C030123:coumarin; D012697:Serine Endopeptidases; D013917:Thrombin; D015951:Factor Xa; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator