Multiplexed protease assays using element-tagged substrates.
Inductively coupled plasma-mass spectrometry (ICP-MS)-based assays lend themselves to multiplexing due to the high resolution between mass channels, the sensitivity, and the reliability of the technique. Here the potential of ICP-MS-based protease assays is demonstrated with a quadruplex assay of cysteine proteases and metalloproteases. Four orthogonal peptide substrates were synthesized for the proteases calpain-1, caspase-3, matrix metalloprotease-9 (MMP-9), and a disintegrin and metalloprotease-10 (ADAM10). Each substrate carries a biotin tag at the C terminus and a diethylenetriaminepentaacetic acid (DTPA)-based lanthanide complex at the N terminus. The results demonstrate that this is a simple and reproducible analysis technique with excellent correlation between the single and multiplex assay formats.
Lathia
Urja S
US
Department of Chemistry, University of Toronto, Ontario, Canada.
Ornatsky
Olga
O
Baranov
Vladimir
V
Nitz
Mark
M
D051722:ADAM Proteins / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D002154:Calpain / Q000737:chemistry / Q000378:metabolism; D053148:Caspase 3 / Q000737:chemistry / Q000378:metabolism; D057056:Cysteine Proteases / Q000737:chemistry* / Q000378:metabolism; D019483:Disintegrins / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry / Q000378:metabolism; D045726:Metalloproteases / Q000737:chemistry* / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry; D013379:Substrate Specificity
D019483:Disintegrins; D001710:Biotin; D019809:Streptavidin; D057056:Cysteine Proteases; D045726:Metalloproteases; D002154:Calpain; D053148:Caspase 3; D051722:ADAM Proteins; D020780:Matrix Metalloproteinase 9