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

Abstract

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 Ͻ0.5 L 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 (R 2 ϭ0.95 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 Ͼ2-fold in AT1aϪ/Ϫ as compared with AT1a ϩ/ϩ . 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.

New Mass Spectrometric Assay for Angiotensin-Converting Enzyme 2 Activity

“…32 The method makes it possible to plot appearances and disappearance of products, reactants, and even intermediates over short reaction times. 24,33 It has the advantage of specificity, speed, and reproducibility. We chose to develop an MS-based assay system because of the ability to use endogenous peptides as substrates and the capacity for direct analysis of the enzymatic peptide products.…”

“…The methodology for tissue measurements is similar to that described previously. 24 The tissues are homogenized on ice in 1:9 (weight/vol) of Tris HCl (50 mmol/L; pH 7.4) containing 2 mmol/L PMSF. The homogenate is centrifuged at 9000g for 10 minutes to remove cellular debris.…”

“…After the incubation period, 1 L of the reaction mixture was spotted onto ProteinChip WCX2 and analyzed as described previously. 24 …”

“…Using endogenous peptide substrates with surface-enhanced laser desorption/ionization time of flight (SELDI-TOF) mass spectrometry (MS) measurements, we were able to show that ACE1 (conversion of Ang I to Ang II) or renin (conversion of tetradecapeptide to Ang I) activity could be measured in small sample volumes (Ͻ1 L plasma). 24 Studies of physiological conditions revealed that there were increases in plasma ACE1 activity in diabetic mice and plasma renin activity in the Ang AT1a receptor deletion model. The key issue behind the methodology is the ability to distinguish and quantify small molecular weight peptides.…”

“…The method was based on our previous work, which established MS as a useful tool for measuring proteolytic enzyme activity, specifically as related to the RAS. 24 The approach uses the natural endogenous substrates for ACE2, Ang I, and Ang II, which are proteolytically cleaved to yield Ang (1-9) and Ang (1-7) respectively. It is anticipated that this MS enzyme assay will have applications in drug screening, antagonist development, and clinical investigations.…”