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.
Baars
M J
MJ
Department of Chemistry, University Plaza, Georgia State University, Atlanta 30303, USA.
Patonay
G
G
D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000032:analysis*; D000818:Animals; D001557:Benzenesulfonates; D019075:Electrophoresis, Capillary / Q000379:methods; D005456:Fluorescent Dyes; D006801:Humans; D007211:Indoles; D007834:Lasers; D009842:Oligopeptides / Q000032:analysis; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D019265:Spectroscopy, Near-Infrared / Q000379:methods