Solid‐phase fluorescent labeling reaction of picomole amounts of insulin in very dilute solutions and their analysis by capillary electrophoresis

Abstract

The fluorescent labeling of peptides at concentrations as low as 10(-8) M can be achieved by using a solid-phase reactor. Using oxidized insulin chain B as a test peptide, we demonstrate the use of an Immobilon CD membrane to capture and preconcentrate peptides. Insulin chain B can then be labeled with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde, while it is still attached to the membrane. Unwanted fluorescent products (attributed to secondary reactions) can be washed away with methanol without significant removal of the labeled insulin chain B, which then can be extracted with a low pH buffer. The analysis by micellar electrokinetic capillary chromatography with post-column laser-induced fluorescence detection (mass limit of detection of 2.4 x 10(-21) moles insulin chain B) results in electropherograms that show great improvement in terms of unwanted peaks and high number of theoretical plates (up to 20 million). The use of the solid-phase reactor allows easy handling of as little as 5 picomoles of insulin chain B.

Capillary electrophoresis hyphenated with UV‐native‐laser induced fluorescence detection (CE/UV‐native‐LIF)

“…A 3 kV run during 5 min released the rest of insulin allowing quantitative measurements of both: the amount of insulin released and its amount remaining in the cell. We can note a method using a solid‐phase 3‐(2‐furoyl)quinoline‐2‐carboxaldehyde labeling reaction of picomole amounts of insulin using CE/LIF 488 nm . These results show how CE/UVLIF can be sensitive compared to visible fluorescence and protein labeling.…”

Laser‐induced fluorescence detection schemes for the analysis of proteins and peptides using capillary electrophoresis

“…Initially it was used to detect picomole amounts of insulin in very dilute solutions using CE-LIF [30]. Recently, the reaction rate, activation energy and LOD of some neurotransmitters with FQ in artificial CSF were studied [31].…”

Protein and peptide analysis by capillary zone electrophoresis and micellar electrokinetic chromatography

“…Although laser-induced fluorescence detection with precolumn derivatization is the most sensitive detection mode in CE, and can even provide a desired selectivity, the incomplete chemical modification at low analyte concentrations may limit its reliability and application. This problem was addressed by using the solid-phase fluorescent labeling reaction by Pinto et al [46]. The labeling of peptides at concentration as low as lo-* M could be achieved by the method.…”