Rapid, sensitive and quantitative assays for proteases are of great significance for drug
development and in diagnosis of diseases. Herein, we describe work towards a novel
assay for the multiplexed detection of proteases using ICP-MS. Protease substrates
were synthesized containing a diethylenetriaminepentaaceticacid(DTPA) ligand to
chelate lanthanide metal ions at the N-terminus, providing a distinct tag for each substrate. A biotin label was appended to the C-terminus allowing separation of uncleaved peptide from the digestion. The enzymatic activities can then be determined by detecting the lanthanide signal of the peptide cleavage products by ICP-MS.
Substrates synthesized include DTPA-Gln-Val-Tyr-Gly-Nle-Nle-Lys(biotin)-amide, DTPA-Asp-Gln-Val-Asp-Gly-Lys(biotin)-amide and DTPA-Gly-Pro-Gln-Gly-Leu-Glu-Ala-Lys-Lys(biotin)-amide for calpain-1, caspase-3 and MMP-9 They were loaded with terbium, holmium and praseodymium respectively. As a proof-of-concept, α-chymotrypsin assays were carried out using DTPA-Asp-Leu-Leu-Val-Tyr-Asp-Lys(Biotin) loaded with lutetium, as a substrate. Calpain-1 assays were also performed. Parallel assays with commercially available fluorogenic substrates for both the enzymes were performed for comparison.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/19310 |
| Date | 04 March 2010 |
| Creators | Lathia, Urja |
| Contributors | Nitz, Mark |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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