Burvenich, Christian

De Spiegeleer, Bart

Pezeshki, Adel

Van Dorpe, Sylvia

Vergote, Valentijn

English

Burvenich, Christian Paul G.

Ghent University Academic Bibliography

INTRODUCTION Inhibiting protease-activity in peptidomics: strategies during sampling and receptor studiesValentijn Vergote1, Adel Pezeshki1,2, Sylvia Van Dorpe1, Christian Burvenich2 and Bart De Spiegeleer1,*1Drug Quality & Registration (DruQuaR) group, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium,                       2Department of Physiology and Biometrics, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.*Corresponding author: bart.despiegeleer@ugent.be (O.Ref.: 2008–546b; Proteomic  Europe Conference 2008, Lisbon, Portugal)DruQuaRDrug Quality & Registration (DruQuaR) format Man06.001.006-v01Most endogenous peptides are prone to fast metabolization by a broad variety of peptidase and protease enzymes that occur in different tissues and cells. As a result, amain problem in peptidomics analysis is peptide degradation during the sampling step, including sampling storage and sample extraction, aiming at high recoveries for allendogenous peptides present in a sample with no (further) breakdown of the proteins and peptides. Related problems are encountered during peptide functional andreceptor binding studies, where protease inhibitors are normally used to improve ligand stability against membrane bound enzymes.EXPERIMENTALA general screening procedure to evaluate peptide metabolic stabilityand protease inhibitor efficacy is described in Figure 1.Sample matricesTypically, plasma, crude tissue/cell homogenates or membrane fractionsare used as prepared for peptidomics analyses and in vitro experimentssuch as radioligand binding and permeability studies. The preparationmay include a protease denaturation/removal treatment such asmicrowave heating.Protease activity testingModel or target peptides are incubated at pH 7.4 in the sample matrixunder examination using a mixing block heater at 37°C. Aliquots arewithdrawn at suitable time points, and analyzed (after acidification,heating and centrifugation) using HPLC with PDA and/or fluorescencedetection. Results are expressed as half-life times, calculated from thecurves obtained.Protease inhibitor efficacy testingPeptides are incubated under the same conditions as above, however inabsence (i.e. reference) and presence of selected protease inhibitors(e.g. EDTA, leupeptin). At a pre-determined time point (e.g. half-lifewithout protease inhibitors), the reaction is stopped and the mixtures areanalyzed as described for protease activity testing. Half-life times arecalculated based upon first order kinetics assumption.RESULTS AND DISCUSSIONCONCLUSIONS• A simple general method can be applied for (a) testing the susceptibility of peptides to peptidase/protease activity in samples (derived from plasma, tissues and cells), (b)verification of enzyme denaturation in sample preparation protocols, and (c) peptidase/protease activity inhibitor efficacy testing.• A general protease inhibitor cocktail and EDTA were found improve the metabolic stability of obestatin in brain homogenates.REFERENCES[1] V. Vergote, S. Van Dorpe, K. Peremans, C. Burvenich and B. De Spiegeleer. In vitro metabolic stability of obestatin: Kinetics and identification of cleavage products. Peptides 29 (2008), pp. 1740-1748.ACKNOWLEDGEMENTS: We thank Nadia Lemeire and Evelyn De Backer for their experimental help.Figure 1. General scheme for peptide stability and protease inhibitor efficacy testingTypical HPLC conditions: Everest C18 238EV54 (250 mm × 4.6 mm, 300 Å, 5 µm) column (Grace Vydac) at 30°C, with (A) 0.1% formic acid in water, and (B) 0.1% formic acid in acetonitrile. The gradient used for the separation was: 95% A + 5% B from 0 to 5 minutes, followed a linear ramp from 5 to 65 min going to 70% A + 30% B. The flow rate was set at 1.0 ml/min [1].Typical results are presented here. Other peptides and sample treatments were successfully analyzed using in the same methodology (data not given).Stability of model peptides upon acidification and heat treatment was demonstrated.The degradation of mouse obestatin in crude mouse brain homogenate () and mouse brain membraneshomogenate (), at a peptide/tissue-protein ratio of 1:10 m/m, is described in Figure 2. It was found thatthis peptide is rapidly degraded when in contact with brain proteases and peptidases. The metabolizationhalf-lives were found to differ by a factor 1.4: 19.3 min and 27.4 min, resp.Results obtained on protease inhibitors are presented in the Table below. Similarly increased half-livesfor both  homogenates were observed for EDTA (metal chelator) and a mixture of six protease inhibitors:4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatin A, E-64, bestatin, leupeptin, and aprotinin.Protease inhibitorHalf-life (min)Crude brain homogenate Brain membranes homogenateUV FL UV FLNone (i.e. reference) 19 19 27 27P8340 Protease Inhibitor Cocktail 52 54 74 77EDTA 38 40 57 67Soybean Trypsin Inhibitor 20 21 37 40APMSF 20 21 15 171,10-Phenanthrolin 24 24 27 29Captopril 20 20 27 30Chymostatin 17 17 28 30 Figure 2. Degradation of mouse obestatin in brain homogenates

Inhibiting protease-activity in peptidomics: strategies during sampling and receptor studies

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Inhibiting protease-activity in peptidomics: strategies during sampling and receptor studies

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