Angiotensinogen (1-14), human

The use of ultraviolet matrix-assisted laser desorption (MALD) to ionize peptides and proteins for analysis in a quadrupole ion trap is described. An ion source was modified to accommodate a fiber optic to transmit laser radiation from a nitrogen laser (337 nm) to the tip of the sample probe containing peptide or protein samples in a matrix of 2,5-dihydroxybenzoic acid (DHB) or 3,4-dimethoxy-4-hydroxy-cinnamic acid. Detection limits are demonstrated with 10 fmol of sperm-whale myoglobin. The dimer of sperm-whale myoglobin was also observed at m/z 34,430. A comparison is made of the tandem mass spectrum of (MS/MS) of human angiotensin I desorbed by MALD to that of the peptide desorbed by liquid secondary-ion mass spectrometry. Both spectra were found to contain abundant structural information.

The development of renin inhibitors for the treatment of hypertension requires highly sensitive substrates to evaluate potency and to characterize the mechanism of tight-binding inhibitors. A series of intramolecularly quenched fluorogenic renin substrates, based on the N-terminal tetradecapeptide sequence of human angiotensinogen (hTDP), was synthesized using a solid-phase technique. Incorporation of the fluorescent amino acid L-Amp [L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid] and the DNP (2,4-dinitrophenyl) group at various positions resulted in >90% quenching efficiency and strong product fluorescence. Shortening the hTDP sequence to an octapeptide from histidine in P5 to histidine in P3' (substrate 3) resulted in an acceptable k(cat)/K(m) (41000 M(-1).s(-1)) and further systematic variation gave substrate 9, DNP-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-L-Amp, with a k(cat)/K(m) value of 350000 M(-1).s(-1) and 94% quenching efficiency. The free side chain of lysine, replacing the isoleucine residue at P6 position in the angiotensinogen sequence, contributed to the increased value for k(cat). The pH dependence of k(cat)/K(m) for renin and substrate 9 showed that the optimal pH is at pH 6-7. It also showed two titrating groups on the acidic side of the pH optimum, and one titrating group with a pK(a) of 7.8 on the alkaline side. The combination of good kinetic and spectroscopic properties resulted in a >20-fold improvement in the sensitivity of renin assay, compared with the commercial substrate Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(DABCYL)-Arg [where EDANS is 5-[(2-aminoethyl)amino]naphthalene-1-sulphonic acid and DABCYL is 4-(4-dimethylaminophenylazo)benzoic acid] (k(cat)/K(m)=268000 M(-1) x s(-1), quenching efficiency <80%). The detection limit in a microplate renin assay was 60 pM, making substrate 9 well suited for the evaluation of inhibitors at picomolar concentrations.

A pore chip protein array (PCPA) concept based on a dual readout configuration, fluorescence imaging, and MALDI-TOF MS has been developed. Highly packed, (>4000 spots/cm2), antibody arrays were dispensed on the porous chip by using a piezo-electric microdispenser. Sandwich assay was made after blocking by addition of a secondary antibody either IgG-FITC-labeled or anti-Ang II. The antigen in the first system was a large protein (IgG), and in the other system, a FITC marked peptide Angiotensin II (Ang II) was used. Ang II antibodies showed specificity for Ang II, while the Ang I antibodies showed binding properties for Ang I, II, and Renin. Fluorescence and MALDI TOF MS read-out was made for IgG and Ang II. A major advantage of the dual read-out PCPA approach is that both affinity binding and mass identity are derived. Detection limits for Ang II on the chip is as low as 500 zmol (Ang II).