title: Development of inductively coupled plasma-mass spectrometry-based protease assays.
abstract: Rapid, sensitive, and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here an assay for protease activity that uses inductively coupled plasma-mass spectrometry (ICP-MS) detection is described. Peptidic alpha-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N terminus to provide a distinct elemental tag. A biotin label was appended to the C terminus of the peptide, allowing separation of uncleaved peptide from the enzymatic digestion. The enzyme activity was determined by quantifying the lanthanide ion signal of the peptide cleavage products by ICP-MS. Biotinylated substrates synthesized include Lu-DTPA-Asp-Leu-Leu-Val-Tyr approximately Asp-Lys(biotin) and Lu-DTPA-betaAla-betaAla-betaAla-betaAla-Gly-Ser-Ala-Tyr approximately Gly-Lys-Arg-Lys(biotin)-amide. Parallel assays with a commercially available fluorogenic substrate (Suc-AAPF-AMC) for alpha-chymotrypsin were performed for comparison. Using the ICP-MS assay, enzyme concentrations as low as 2pM could be readily detected, superior to the detection limit of an assay using the alpha-chymotrypsin fluorogenic substrate (Suc-AAPF-AMC). Furthermore, we demonstrated the use of this approach to detect chymotrypsin activity in HeLa cell lysates.
authors.affiliation: Department of Chemistry, University of Toronto, Toronto, Ont., Canada M5S 3H6.
authors.forename: Urja S
authors.initials: US
authors.lastname: Lathia
authors.forename: Olga
authors.initials: O
authors.lastname: Ornatsky
authors.forename: Vladimir
authors.initials: V
authors.lastname: Baranov
authors.forename: Mark
authors.initials: M
authors.lastname: Nitz
mesh_terms: D000595:Amino Acid Sequence; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002918:Chymotrypsin / Q000032:analysis* / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D028581:Lanthanoid Series Elements / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D028581:Lanthanoid Series Elements; D001710:Biotin; D002918:Chymotrypsin; C117608:alpha-chymotrypsin