Ultrasensitive Detection of Closely Related Angiotensin I Peptides Using Capillary Electrophoresis with Near-Infrared Laser-Induced Fluorescence Detection

A novel near-infrared (NIR) fluorescent dye (NN382, LICOR, Inc.) was evaluated as an ultrasensitive peptide-labeling reagent for use with capillary electrophoresis (CE). Six angiotensin I (Ang-I) variants were selected as model peptides for the derivatization and separation studies. The closely related decapeptides were labeled with the NIR dye, separated using CE, and detected by NIR laser-induced fluorescence. Derivatization of the peptides was achieved under aqueous conditions using 2.5-500 pmol of Ang-I in a 50-microL sample (5 x 10(-8)-1 x 10(-5)M), and between 1.3 and 254 amol of the labeled peptides were injected on column. The fluorescence response was linear over a 200-fold range (correlation r > or = 0.9986). The limit of detection (SNR = 3, signal/RMS noise) ranged from 100 to 300 zmol, for the six Ang-I variants. Four of six peptides were resolved from each other and excess dye using capillary zone electrophoresis with a simple 50 mM phosphate run buffer, pH 7.2. Two pairs of coeluting peptides were successfully resolved using micellar electrokinetic chromatography with a nonionic surfactant, Triton X-100. The NIR amine-labeling reagent NN382 is a viable alternative to using visible fluorophores for CE methods requiring high sensitivity.