Item – Theses Canada

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OCLC number
796917424
Link(s) to full text
LAC copy
LAC copy
Author
Lathia, Urja,1984-
Title
Development of lanthanide-tagged substrates towards detection of proteases by inductively coupled plasma-mass spectrometry (ICP-MS).
Degree
M. Sc. -- University of Toronto, 2008
Publisher
Ottawa : Library and Archives Canada = Bibliothèque et Archives Canada, [2011]
Description
1 microfiche
Notes
Includes bibliographical references.
Abstract
<?Pub Inc> 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 diethylenetriaminepentaacetic acid (DTPA) ligand to chelate lanthanide metal ions at the N-terminus, providing a distinct tag for each substrate when complexed with a lanthanide metal. A biotin label was appended to the C-terminus allowing separation of uncleaved peptide from the digestion. The enzymatic activities can then be determined in a multiplexed fashion by detecting the lanthanide signal of the peptide cleavage products by ICP-MS. Biotinylated substrates synthesized include DTPA-Gln-Val-Tyr-Gly-Nle-Nle-Lys(biotin)-amide for calpain-1, DTPA-Asp-Gln-Val-Asp-Gly-Lys(biotin)-amide for caspase-3 and DTPA-Gly-Pro-Gln-Gly-Leu-Glu-Ala-Lys-Lys(biotin)-amide for MMP-9. The substrates were loaded with terbium, holmium and praseodymium respectively. As a proof of concept, [alpha]-chymotrypsin assays was 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.
ISBN
9780494675038
0494675039