Ultrasensitive detection of closely related angiotensin I peptides doing capillary electrophoresis with near-infrared laser-induced fluorescence detection

A novel near-infrared (NIR) fluorescent dye (NN382, LICOR, Inc.) was evalua ted as an ultrasensitive peptide-labeling reagent for use with capillary el ectrophoresis (CE). Six angiotensin I (Ang-I) variants were selected as mod el peptides for the derivatization and separation studies. The closely rela ted decapeptides were labeled with the NIR dye, separated using CE, and det ected 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-mu L sample (5 x 10(-8)-1 x 10(-5) M), and between 1.3 and 254 amol of the labe led peptides were injected on column. The fluorescence response was linear over a 200-fold range (correlation r greater than or equal to 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 ot her and excess dye using capillary zone electrophoresis with a simple 50 mM phosphate run buffer, pH 7.2. Two pairs of coeluting peptides were success fully 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 se nsitivity.