Proteolytic processing of angiotensin-I in human blood plasma.

In mammalian species, except humans, N-terminal processing of the precursor peptide angiotensin I (ANG-1-10) into ANG-2-10 or ANG-3-10 was reported. Here we hypothesize that aminopeptidase-generated angiotensins bearing the same C-terminus as ANG-1-10 are also present in humans. We demonstrate the time dependent generation of ANG-2-10, ANG-3-10, ANG-4-10, ANG-5-10 and ANG-6-10 from the precursor ANG-1-10 by human plasma proteins. The endogenous presence of ANG-4-10, ANG-5-10 and ANG-6-10 in human plasma was confirmed by an immuno-fluorescence assay. Generation of ANG-2-10, ANG-3-10 and ANG-4-10 from ANG-1-10 by immobilized human plasma proteins was sensitive to the cysteine/serine protease inhibitor antipain. The metal ion chelator EDTA inhibited Ang-6-10-generation. Incubation of the substrates ANG-3-10, ANG-4-10 and ANG-5-10 with recombinant aminopeptidase N (APN) resulted in a successive N-terminal processing, finally releasing ANG-6-10 as a stable end product, demonstrating a high similarity concerning the processing pattern of the angiotensin peptides compared to the angiotensin generating activity in plasma. Recombinant ACE-1 hydrolyzed the peptides ANG-2-10, ANG-3-10, ANG-4-10 and ANG-5-10 into ANG-2-8, ANG-3-8, ANG-4-8 and ANG-5-8. Since ANG-2-10 was processed into ANG-2-8, ANG-4-8 and ANG-5-8 by plasma proteases the angiotensin peptides bearing the same C-terminus as ANG-1-10 likely have a precursor function in human plasma. Our results confirm the hypothesis of aminopeptidase mediated processing of ANG-1-10 in humans. We show the existence of an aminopeptidase mediated pathway in humans that bypasses the known ANG-1-8-carboxypeptidase pathway. This expands the knowledge about the known human renin angiotensin system, showing how efficiently the precursor ANG-1-10 is used by nature.

Noncovalent antibody immobilization on porous silicon combined with miniaturized solid-phase extraction (SPE) for array based immunoMALDI assays.

This paper presents a new strategy to combine the power of antibody based capturing of target species in complex samples with the benefits of microfluidic reverse phase sample preparation on an integrated sample enrichment target (RP-ISET) and the analysis speed of MALDI MS. The immunoaffinity step is performed on an in-house developed 3D-structured high surface area porous silicon (PSi) matrix, which allows efficient antibody immobilization by surface adsorption without any coupling agents in 30-60 min. The hydrophilic nature of the porous silicon surface at the molecular level displays a low adsorption of background peptides when exposed to complex digests or plasma samples, improving the conditions for the antigen specific extraction and subsequent readout. At the same time, the hydrophobic behavior, due to the nanostructured surface, of the PSi material facilitates liquid confinement during the assay. Using a footprint conforming to the standard for 384 well microplates, direct adaption of the protocol into standard sample handling robots is possible. The performance of the proposed immunoaffinity PSi-ISET immunoMALDI (iMALDI) assay was evaluated by specific detection of angiotensin I at a 10 femtomol level in diluted plasma samples (10 µL, 1 nM).

Rapid MALDI mass spectrometric analysis with prestructured membrane filters and functionalized diamond nanocrystals.

A solid-phase extraction method based on prestructured membrane filters and functionalized diamond nanocrystals is developed to facilitate analysis of biopolymers with MALDI mass spectrometry. The method integrates isolation, purification, and concentration in one step and is adaptable to high-throughput peptide fingerprint mapping of intact proteins and enzymatic digests. The feasibility of the method was demonstrated using diamond nanocrystals surface-derivatized with carboxylate groups and subsequently coated with poly-lysines. These nanocrystals, which are capable of extracting proteins from highly dilute solutions contaminated with salts and detergents, can be easily isolated by filtration through paraffin-coated PVDF membrane filters pre-patterned with channels for direct MALDI mass spectrometric analysis. This method is facile, easy to operate, and the analysis of each sample solution (with a volume up to 500 microL) can be completed in 10 min. With this method, it is possible to achieve high-throughput analysis of proteins by preparing an array of filtration spots on paraffin-coated PVDF membrane that matches the MALDI sample target of the commercial mass spectrometer.

Angiotensin I retention behavior on Butyl-Sepharose under linear loading chromatographic conditions.

Adsorption behavior of angiotensin I on a commercial Butyl-Sepharose support has been studied in function of temperature and ammonium sulphate concentration. Under isocratic elution conditions and at the higher salt concentrations, a characteristic of the chromatographic performance of angiotensin I was the broadness of the corresponding peak and in most of the cases the appearance of two peaks. These results have been interpreted in terms of on-column cis-trans isomerization of angiotensin I (a proline containing polypeptide) followed by its "re-conformation" after the interaction with the support. It has been proposed that the peak splitting phenomenon, a combined effect between temperature, salt concentration in the mobile phase and the ligand, is caused by slow kinetics of isomerization that is on the same time scale as the chromatographic separation. Salt concentration and temperature promote the conversion of the trans form of angiotensin I into its cis form, which has a bigger hydrophobic surface area, in the presence of Butyl-Sepharose. The retention of the cis form of angiotensin I increases with the increase in salt concentration in the mobile phase and seems to be little affected by temperature.

Iron protoporphyrin-modified monolithic support for on-line preconcentration of angiotensin prior to CE analysis.

An on-line preconcentration method using a polymeric monolithic support is proposed for the retention of the decapeptide angiotensin I and its subsequent analysis by CZE. Monolithic capillary columns were prepared in fused-silica (FS) capillaries of 150 microm id by ionizing radiation-initiated in situ polymerization and cross-linking of diethylene glycol dimethacrylate and glycidyl methacrylate, and chemically modified with iron protoporphyrin IX (Fe-ProP). Monolithic microcolumns (8 mm long) were coupled on-line to the inlet of the separation capillary (FS capillary, 75 microm id x10 cm from the inlet to the microcolumn and 27 cm from the microcolumn to the detector). Angiotensin I was released from the sorbent by a 50 mM sodium phosphate, pH 2.5/ACN, 75:25 v/v solution and then analyzed by CZE with UV absorption detection at 214 nm. The concentration LOQ (CLOQ) was 0.5 ng/mL. The Fe-ProP-derivatized monolithic microcolumn coupled to the separation capillary exhibited a high retention capacity for peptide angiotensin I, and showed as much as 10,000-fold improvement in concentration sensitivity.

Ultra-rapid separation of an angiotensin mixture in nanochannels using shear-driven chromatography.

The present paper reports on the separation of a mixture of fluorescein isothiocyanate-labeled angiotensin I and II peptides in a shear-driven nanochannel with a C18-coating and using an eluent consisting of 5% acetonitrile in 0.02 M aqueous phosphate buffer at pH 6.5. The flat-rectangular nanochannel in fused silica consisted of an etched structure in combination with a flat moving wall. The very fast separation kinetics that can be achieved in a nanochannel allowed to separate the angiotensin peptides in less then 0.2 s in a distance of only 1.8 mm. Plate heights as small as 0.4 microm were calculated after substraction of the injection effect.

High-performance SPME/AP MALDI system for high-throughput sampling and determination of peptides.

This paper presents the performance characteristics for a new multiplexed solid-phase microextraction/atmospheric pressure matrix-assisted laser desorption/ionization (SPME/AP MALDI) source configuration for a hybrid quadrupole-linear ion trap instrument. The results demonstrate that thorough optimization of parameters such as SPME coating material, optics configurations, extraction solvents, and fiber capacity provides dramatic sensitivity improvements (>1000x) over previous reports in the literature. The multiplexed SPME plate is capable of simultaneous extraction from 16 different wells on a multiwell plate, eliminating the need for extensive sample preparation. Subfemtomole sensitivity is demonstrated for peptide standards and protein digests with run-run reproducibility ranging from approximately 13 to 31%. This high-performance SPME/AP MALDI system shows potential for high-throughput extraction from biological samples.

Dynamic pH junction technique for on-line preconcentration of peptides in capillary electrophoresis.

A method based on the presence of a dynamic pH junction within the capillary to induce band narrowing for enhanced detection sensitivity for some peptides is presented. This technique is predicated on a sharp reduction in an analyte's migration velocity following a reversal of its electrophoretic direction from the acidic sample zone to the basic BGS zone. Larger-than-usual injection volumes of samples in relatively high-conductivity matrices were enabled, without degrading peak shape, resolution and efficiency. The size of the original sample plug was reduced by as much as 38-fold, and improvement in detector response in terms of peak height by as much as 124-fold was obtained. The effects of pH and concentration of the sample matrix, and the length of sample injection on the efficiency of the technique are discussed.

Identification of angiotensin I in a cyclostome, Lampetra fluviatilis.

Angiotensin I (ANG I) was isolated from incubates of plasma and kidney extracts of the river lamprey, Lampetra fluviatilis, using eel vasopressor activity as an assay during purification. Its sequence was Asn-Arg-Val-Tyr-Val-His-Pro-Phe-Thr-Leu as determined by the sequence analysis and mass spectrometry. The sequence was confirmed by identity of the elution profile with the synthetic peptide in two different reverse-phase columns of high-performance liquid chromatography. Lamprey ANG I produced dorsal-aortic pressor responses in L. fluviatilis but the rise was very small in comparison to that produced by angiotensin II. Angiotensin III produced an even bigger increase. It was not possible to demonstrate a difference in response to Asn(1) (lamprey) ANG I and Asp(1) (human) ANG I. The present study directly demonstrated the presence and biological activity of the renin-angiotensin system in the most primitive extant vertebrates, the cyclostomes. Thus the renin-angiotensin system is a phylogenetically old hormonal system that is present throughout the vertebrates.

Isolation, synthesis, and biological activity of flounder [Asn1,Ile5,Thr9] angiotensin I.

A novel angiotensin I (ANG I) has been isolated from incubates of plasma and kidney extracts of the flounder, Platichthys flesus, using ion-exchange, gel-permeation, and reverse-phase high performance liquid chromatography (HPLC). Its sequence was determined as H-Asn-Arg-Val-Tyr-Ile-His-Pro-Phe-Thr-Leu-OH by sequence analysis and mass spectrometry. No vasopressor activity was detected at the elution position of [Asp(1)] ANG I in ion-exchange HPLC. The sequence was confirmed by identity of the elution position with the synthetic peptide in two different HPLC systems. When compared with ANG I isolated from other teleost fish, flounder ANG I uniquely has an isoleucine at position 5 rather than valine. Injection of angiotensin II (ANG II) into chronically cannulated flounder resulted in a dose-dependent pressor response, native [Asn(1),Ile(5)] ANG II, was found to elicit pressor responses comparable with those seen when teleost [Asn(1),Val(5)] ANG II and human [Asp(1),Ile(5)] ANG II were injected into flounder over the dose range 0.02-1.00 nmol/kg(-1). Plasma concentrations of the neurohypophysial peptide AVT were measured in chronically cannulated flounder following the injection of ANG II to examine the effect of ANG II on circulating AVT concentration. The injection of [Asn(1),Ile(5)] ANG II (1 nmolkg(-1)) or [Asp(1),Ile(5)] ANG II (2.5 nmolkg(-1)) resulted in a significant fall in the circulating levels of AVT suggesting that ANG II either directly or indirectly negatively influences AVT secretion.

Integration of solid-phase extraction membranes for sample multiplexing: application to rapid protein identification from gel-isolated protein extracts.

The present report describes the design and application of a dual sprayer system for high-throughput proteome analysis. This system comprises parallel solid-phase extraction cartridges used for preconcentration and desalting of proteolytic digests prior to nanoelectrospray mass spectrometry analyses. Tryptic peptides from in-gel digest of protein bands/spots are first adsorbed on styrene divinyl benzene membrane and subsequently eluted with a short plug of organic buffer prior to infusion to the mass spectrometer at a flow rate of typically 500 nL/min. Tryptic peptide eluting from the membrane are analyzed by the mass spectrometer by moving in turn each sprayer in front of the sampling orifice. Sequential injection, preconcentration and analyses of tryptic digests are typically achieved with a throughput of up to 3.5 min/sample and a detection limit of approximately 8-80 fmol per injection. Replicate injections of peptide mixtures indicated that reproducibility of peak areas ranged from relative standard deviations (RSD) of 1.1% to 4.5%. The application of this device is demonstrated for digests of gel-isolated proteins obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of rat liver plasma membrane and from two-dimensional gel electrophoresis of total cell lysate extracts from human prostatic cancer cell.

Separation and detection of angiotensin peptides by Cu(II) complexation and capillary electrophoresis with UV and electrochemical detection.

The use of capillary electrophoresis (CE) with on-capillary Cu(II) complexation for the determination of angiotensin and its metabolites is described. The resulting copper-peptide complexes can be detected using either UV or electrochemical (EC) detection. Optimal reaction and separation conditions for the angiotensin peptides were first determined using CE with UV detection. With UV detection, the limit of detection (signal-to noise ratio S/N = 3) for native angiotensin II was 18 microM, while the limit of detection (LOD) obtained for the copper-angiotensin II complex is 2 microM. CE with EC detection was then evaluated, yielding significantly lower LODs--2 microM for native angiotensin II and 200 nM for the copper-angiotensin II complex. The addition of copper to the run buffer improved the separation and sensitivity for both CE-UV and CE-EC detection. The method was demonstrated by monitoring the conversion of angiotensin I to angiotensin II in plasma via angiotensin-converting enzyme (ACE) and subsequent inhibition of ACE by captopril.

[Study on the determination of peptide mixture by HPCE].

A rapid method for the simultaneous assay of 7 peptide mixture, including angiotensin I, II, III, substance P, neurokinin, somatostatin and neurotensin, by high performance capillary electrophoresis has been established. The nature, pH and concentration of buffer, running voltage, detection wavelength, injection time and the effective length of amino-coated capillary were defined with the results of experiment. With 50 mmol/L ammonium acetate (pH 4.5) as running buffer and siphonage injection for 10 seconds, the measurements were carried out at 25 degrees C and 10 kV running voltage [(-)-->(+)] applied to a 57 cm x 75 microns i.d. (50 cm effective length) amino-coated capillary. The 7 peptide mixture was determined by a UV detector at 214 nm. The total time for separation and determination was within 8 min. The recoveries ranged from 95% to 98% with RSD from 2.9% to 4.2%. It has been found that the 75 microns i.d. capillary has higher sensitivity than 50 microns, but its efficiency and Rs were worse.

Cyclodextrin aided separation of peptides and proteins by capillary zone electrophoresis.

Carboxymethylated-beta-cyclodextrin (CMBCD) in the electrophoretic medium (aqueous 50 mM sodium phosphate, pH 2.5) enhanced the separation using raw fused-silica capillaries in CZE of the four standard proteins: alpha-chymotrypsinogen A, cytochrome c, lysozyme and ribonuclease A. Furthermore, with 20 mM CMBCD in the electrophoretic medium, the cis-trans isomers of angiotensin could be separated at room temperature, whereas the separation of the conformers required subambient temperatures as low as -20 degrees C without CMBCD in the electrophoretic medium [50 mM sodium phosphate (pH 2.5), containing 10% (v/v) methanol]. Addition of heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DMBCD) had no effect on the separation of the above proteins and peptides. The results suggest that in microcolumn separation techniques, certain cyclodextrin additives can be useful selectivity enhancers.

Adrenal, kidney, and heart angiotensins in female murine Ren-2 transfected hypertensive rats.

We analyzed by high-performance liquid chromatography and radioimmunoassay angiotensin I (Ang I), Ang II, Ang-(1-7), and metabolites in the adrenal, kidney and heart of normotensive female Sprague-Dawley (SD) and transgenic hypertensive [TGR(mRen-2)27] rats carrying the murine Ren-2d renin gene. The monogenetic model of hypertensive rats had significant increases in adrenal Ang II; whereas in the kidney Ang II was unchanged, but Ang I and Ang-(1-7) were significantly lower. Cardiac Ang I, Ang II, and Ang-(2-10) were significantly reduced in transgenic rats, while Ang-(2-7) was increased. In SD and transgenic rats kidney and adrenal angiotensins increased primarily during estrus or proestrus. In female transgenic rats the increased adrenal Ang II and the sustained renal Ang II may contribute to the established phase of hypertension.

Separation of proteins and peptides by capillary electrochromatography in diol- and octadecyl-modified etched capillaries.

This study involves the evaluation of a capillary electrochromatography method based on etching the inner walls of a fused-silica tube, which is subsequently modified by a silanization/hydrosilation reaction scheme. Two different organic moieties, octadecyl and diol, are attached to the etched capillary wall. The performance of these two columns is compared to a bare capillary using peptide (angiotensins) and protein samples. It is concluded that the etching process increases the surface area of the inner wall sufficiently to induce solute-bonded phase interactions for the capillaries modified with the octadecyl and diol moieties. The separation capabilities of the two modified capillaries are not the same, presumably due to differences in the chemical properties of the two ligands. When compared to a bare capillary where separation is due only to electrophoretic mobility effects, the bonded etched capillaries also exhibit significant differences in separation factors for the same solutes under identical experimental conditions.

Spontaneous inhibition or inactivation of renin in rat plasma.

Renin appears to be rapidly inactivated in vitro. The present study was undertaken to clarify this observation and to establish the existence of substances involved in renin inactivation. The disappearance rate of renin (including pure renin) was measured in plasma incubated at 37 degrees C and in circulating blood. Pure renin added to plasmas disappears in vitro at the same rate (t1/2 approximately or equal to 40 min) that renin in plasma from normal rats and from rats submitted to a hemorrhage. This process appears not to be mediated by proteases. The disappearance rate of endogenous renin in the normal group (n = 18) was 39.7 min with rapid phase (R) of t1/2 = 14.2 min and a slow phase (S) of t1/2 = 94.3 min), whereas it was 32.1 min (t1/2 R = 13.1 min and t1/2 S = 69.1 min) in rats submitted to an hemorrhage (n = 6). The t1/2 of pure renin was 31.4 min (t1/2 R = 13.3 min and t1/2 S = 69.2 min). Incubation of plasma reveals that renin is inactivated or inhibited in vitro at a similar rate than in circulating plasma. These results suggest that inactivation and sequestration of renin could be two independent mechanisms in the maintenance of plasma renin activity.

Angiotensin II formation in the intact human heart. Predominance of the angiotensin-converting enzyme pathway.

It has been proposed that the contribution of myocardial tissue angiotensin converting enzyme (ACE) to angiotensin II (Ang II) formation in the human heart is low compared with non-ACE pathways. However, little is known about the actual in vivo contribution of these pathways to Ang II formation in the human heart. To examine angiotensin II formation in the intact human heart, we administered intracoronary 123I-labeled angiotensin I (Ang I) with and without intracoronary enalaprilat to orthotopic heart transplant recipients. The fractional conversion of Ang I to Ang II, calculated after separation of angiotensin peptides by HPLC, was 0.415 +/- 0.104 (n = 5, mean +/- SD). Enalaprilat reduced fractional conversion by 89%, to a value of 0.044 +/- 0.053 (n = 4, P = 0.002). In a separate study of explanted hearts, a newly developed in vitro Ang II-forming assay was used to examine cardiac tissue ACE activity independent of circulating components. ACE activity in solubilized left ventricular membrane preparations from failing hearts was 49.6 +/- 5.3 fmol 125I-Ang II formed per minute per milligram of protein (n = 8, +/- SE), and 35.9 +/- 4.8 fmol/min/mg from nonfailing human hearts (n = 7, P = 0.08). In the presence of 1 microM enalaprilat, ACE activity was reduced by 85%, to 7.3 +/- 1.4 fmol/min/mg in the failing group and to 4.6 +/- 1.3 fmol/min/mg in the nonfailing group (P < 0.001). We conclude that the predominant pathway for angiotensin II formation in the human heart is through ACE.

A novel angiotensin I isolated from an elasmobranch fish.

It is believed that the renin-angiotensin system evolved initially in primitive bony fishes and is absent from elasmobranchs. We have isolated angiotensin I from the incubates of plasma and kidney extracts of an elasmobranch fish, Triakis scyllia, using eel vasopressor activity as an assay system. Its sequence was determined to be H-Asn-Arg-Pro-Tyr-Ile-His-Pro-Phe-Gln-Leu-OH. Dogfish angiotensin I is teleost-like because of an asparagine residue at position 1 but it is mammalian-like because of an isoleucine residue at position 5. The unique and most important substitution in dogfish angiotensin I is a proline residue at position 3 which may cause significant changes in its tertiary structure. A glutamine residue at position 9 is also unique among all angiotensin Is sequenced to date. Dogfish angiotensin I is more potent than rat angiotensin I in its vasopressor activity in the dogfish but the relationship is reversed in the rat. Thus angiotensin receptors as well as the hormone molecules appear to have evolved during vertebrate phylogeny. Our findings establish the elasmobranch renin-angiotensin system and support the hypothesis that the renin-angiotensin system is a phylogenetically old hormonal system which plays important roles in cardiovascular and fluid homeostasis.