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.