In situ and multisubstrate detection of elastase enzymatic activity external to microdialysis sampling probes using LC-ESI-MS.
Extracellular proteases play significant roles in mammalian development and disease. Enzymatic activity external to a microdialysis sampling probe can be determined by infusing judicious choices of substrates followed by collecting and measuring the products. Porcine pancreatic elastase was used as a model enzyme with two substrates possessing different cleavage sites, N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin (FL-substrate) and N-succinyl-Ala-Ala-Ala-p-nitroanilide (UV-substrate). These substrates were infused through the microdialysis sampling probe to a solution containing elastase. The resulting four products and the remaining two substrates were collected into the dialysate and were subsequently analyzed off-line using liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). All analytes were identified using extracted ion chromatograms of m/z 628 (FL-substrate), m/z 452 (UV-substrate), m/z 471 (N-methoxysuccinyl-Ala-Ala-Pro-Val, FL-NTP), m/z 332 (N-succinyl-Ala-Ala-Ala, UV-NTP), m/z 176 (7-amino-4-methylcoumarin, AMC), and m/z 139 (p-nitroaniline, pNA). FL-NTP and FL-substrate exhibited 10-fold higher ion production as compared to AMC with equimolar standards. Microdialysis sampling combined with LC-ESI-MS detection allowed for in situ determination of the enzymatic activity of a protease external to the microdialysis probe when using different peptide-based substrates.
Wang
Ying
Y
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Zagorevski
Dmitri V
DV
Stenken
Julie A
JA
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017551:Microdialysis; D010196:Pancreatic Elastase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013379:Substrate Specificity
D010196:Pancreatic Elastase