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.

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.