Published Online
on August 15, 2005

A more recent version of this article appeared on October 1, 2005

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Submitted on April 28, 2005
Revised on May 23, 2005

Novel Mass Spectrometric Methods for Evaluation of Plasma Angiotensin Converting Enzyme 1 and Renin Activity

Khalid M. Elased^*; David R. Cool; and Mariana Morris

From Wright State University School of Medicine, Department of Pharmacology and Toxicology, Dayton, Ohio.

^* To whom correspondence should be addressed. E-mail: Khalid.elased{at}wright.edu.

Abstract--This article demonstrates the applicability of quantitative proteomics to assays of proteolytic enzyme activity. A novel assay was developed for measurement of renin and angiotensin-converting enzyme (ACE) activity in plasma. The method was validated in animal models associated with alterations of the renin angiotensin system (RAS). Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) with a ProteinChip Array technology, plasma renin and ACE1 could be measured in <0.5 µL of plasma. Plasma is incubated with peptide substrates for renin and ACE, tetradecapeptide (TDP), and angiotensin I (Ang I), respectively. The reactions mixtures are spotted onto the ProteinChip WCX2 and detected using SELDI-TOF-MS. Peak height or area under curve for TDP, Ang I, and angiotensin II (Ang II) peaks are measured. There was a linear relationship between disappearance of substrate and appearance of products for both renin and ACE (R²=0.95 to 0.98). ACE1 activity was blocked with chelating agents (EDTA and 1,10 phenanthrolene), indicating action of a metalloprotease. The ACE1 inhibitor, captopril, selectively blocked ACE1. Renin activity was specifically blocked with renin inhibitor and was not affected by phenanthrolene or captopril. Animal models tested were Ang AT1a receptor-deficient and streptozotocin (STZ) diabetic mice. Plasma renin activity was increased >2-fold in AT1a^-/- as compared with AT1a^+/+. In STZ diabetic mice, ACE1 was increased 2-fold as compared with controls. The advantage of the method is that it is tagless, does not require additional purification steps, and is extremely sensitive. The approach can be multiplexed and used for identification of novel substrates/inhibitors of the RAS.

Key words: angiotensin • angiotensin-converting enzyme • captopril • hypertension • renin

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