Assessing protease activity pattern by means of multiple substrate ESI-MS assays.
The development of a simultaneous multiple substrate enzymatic assay based on electrospray ionization mass spectrometry (ESI-MS) detection is described. This multiplexing assay scheme was employed in a parallel proteolytic enzyme activity screening. As model systems, the respective activities of trypsin, thrombin, chymotrypsin, bromelain, ficin and elastase towards seven different substrates were assessed. The resulting activity patterns were evaluated semi-quantitatively ranking the enzymatic activities in five classes of activity (very high, high, medium, low and no activity) with respect to the individual substrates. The validity of the MS-based multiplexing assay scheme was proved by comparison with the results obtained from single substrate assays detected by means of UV/vis absorption at 405 nm, showing good agreement of the resulting activity patterns and classifications.
Liesener
André
A
University of Twente, Department of Chemical Analysis, P. O. Box 217, 7500 AE Enschede, The Netherlands.
Karst
Uwe
U
D017022:Flow Injection Analysis / Q000295:instrumentation; D010447:Peptide Hydrolases / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013379:Substrate Specificity
D010447:Peptide Hydrolases