Ultrasensitive detection of closely related angiotensin I peptides doing capillary electrophoresis with near-infrared laser-induced fluorescence detection
Mj. Baars et G. Patonay, Ultrasensitive detection of closely related angiotensin I peptides doing capillary electrophoresis with near-infrared laser-induced fluorescence detection, ANALYT CHEM, 71(3), 1999, pp. 667-671
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.