title: A method for rapid protease substrate evaluation and optimization.
abstract: We have developed a high throughput assay for the measurement of protease activity in solution. This technology will accelerate research in functional proteomics and enable biologists to streamline protease substrate evaluation and optimization. The peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety and a fluorescent tag is attached to the amino terminus. Protease cleavage causes the biotin containing fragment to be detached from the labeled peptide fragment. Following the protease treatment, all biotin containing species (uncleaved substrates and the cleaved carboxy terminal fragment of the substrate) are removed by incubation with streptavidin beads. The cleaved fluorescently labeled amino terminal part of the substrate remains in solution. The measured fluorescence intensity of the solution is directly proportional to the activity of the protease. This assay was validated using trypsin, chymotrypsin, caspase-3, subtilisin-A, enterokinase and tobacco etch virus protease.
authors.affiliation: Illumina, Inc., 9885 Towne Centre Drive, San Diego, CA 92121-1975, USA.
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kozlov
authors.forename: Peter C
authors.initials: PC
authors.lastname: Melnyk
authors.forename: Chanfeng
authors.initials: C
authors.lastname: Zhao
authors.forename: John P
authors.initials: JP
authors.lastname: Hachmann
authors.forename: Veronika
authors.initials: V
authors.lastname: Shevchenko
authors.forename: Anu
authors.initials: A
authors.lastname: Srinivasan
authors.forename: David L
authors.initials: DL
authors.lastname: Barker
authors.forename: Michal
authors.initials: M
authors.lastname: Lebl
mesh_terms: D001681:Biological Assay / Q000379:methods*; D020169:Caspases / Q000737:chemistry / Q000235:genetics; D015394:Molecular Structure; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D012996:Solutions / Q000737:chemistry; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D010455:Peptides; D012996:Solutions; D010447:Peptide Hydrolases; D020169:Caspases