Angiotensin 1

Angiotensin I (Bachem, Torrance, CA, USA) was diluted to 100 μM in PBS and reacted with 200 μM heavy or light ONOO⁻ for 15 min at 37 °C. Reactions were quenched by the addition of formic acid to a final composition of 5%. In a total volume of 100 μL, light and heavy Angiotensin I nitration reactions were combined to generate mixtures of 1:1, 0.5:1 and 0.25:1 ratios of light:heavy nitro-Angiotensin I. All dilutions were desalted in C18 SPE columns (The Nest Group, Southborough, MA, USA) and eluted in 90:10 acetonitrile:HPLC-grade water (0.1% formic acid). Desalted samples were concentrated under vacuum at 50 °C until dried out completely. Light and heavy nitrated Angiotensin I mixtures were resuspended to 100 μM total Angiotensin I (assuming 100% recovery) in HPLC-grade 0.1% formic acid in water.

The following synthetic analogues of human bioactive peptides were analyzed: angiotensin II (Sigma, catalog number A9525), angiotensin I, bradykinin, luteinizing-hormone-releasing hormone (LHRH), α-melanocyte-stimulating hormone (α-MSH), neurotensin, oxytocin, substance P, and vassopresin (all Bachem, catalog numbers H-1680, H-1970, H-6728, H-1075, H-4435, H-2510, H-1890 and H-1780, respectively). Stock solutions of peptides (10 mM) were prepared in water. rSmPOP (0.7 μg) was incubated at 37°C for 16 h with 25 nmol of peptide in 0.1 M Tris-HCl, pH 8.0, in a total volume of 50 μL. The reaction was stopped by adding TFA to a final concentration of 1%. The resulting fragments were purified by RP-HPLC over a C18 column (Vydac, 25 x 0.46 cm) using a TFA/acetonitrile system and characterized by electrospray ionization mass spectrometry on an LCQ Classic Finnigan Mat device (Thermo Finnigan).