Investigation of acridinium labelling for chemiluminescence detection of peptides separated by capillary electrophoresis

Abstract

Abstract. An acridinium ester has been used to tag mixtures of peptides followed by separation by capillary electrophoresis with chemiluminescence detection. The labelling of the peptides was performed at the picomole level. The chemiluminescence reaction conditions were optimized for the acridinium tag resulting in CE separations performed on attomole amounts of tagged peptides. Baseline resolution was obtained for a separation of standard peptides. The detection sensitivity was found to improve when multiple acridinium labels were attached to the peptides. The effect of acridinium tagging on the migration rate of the peptides was also studied. The increased weight of the acridinium tagged analyte increased the run time and resolution of a separated tryptic digest without resulting in excessive band broadening.

Capillary electrophoresis chemiluminescent detection system equipped with a two‐step postcolumn flow interface for detection of some enkephalin‐related peptides labeled with acridinium ester

“…The interface designed for a CE-CL system to detect acridinium has been reported by Ruberto and Grayeski [14,15]. Their interface could only be suitable for an electrophoretic buffer whose pH value was less than 3.0, and the higher pH value of electrophoretic buffer would cause severe loss of CL intensity due to the hydrolysis of acridinium esters.…”

“…Acridinium compounds seem to be the good reagents for CE-CL owing to their highly CL quantum yield and rapid CL reaction. Ruberto and Grayeski [14,15] developed a detection interface for the postcolumn addition of acridinium ester in a CE system to evaluate the efficiency of this CL reaction. However, acridinium esters were easily hydrolyzed to give their pseudo-base form above pH 3, and the hydrolyzation of acridinium esters would decrease the CL signals by more than 99% if the pH was higher than 4 [14].…”

Trends towards sensitive detection in capillary electrophoresis: an overview of some recent developments

“…Also, the positive charge of the quaternary nitrogen atom in the ring structure provides greater mobility in the applied electric fields. Using a synthetic acridinium ester (4-(2-succinimidyloxycarbonylethyl)phenyl-10-methyl-acridinium-9-carboxylate fluorosulfonate) as a tag, this CL detection was satisfactorily applied to the separation of attomole quantities of peptides by CE (Ruberto and Grayeski, 1994).…”

Simultaneous determination of isoniazid and p‐aminosalicylic acid by capillary electrophoresis using chemiluminescence detection

“…Thus, several CL systems including luminol (24−27), peroxyoxalate (28), acridinium (29,30), tris (2,2-bipyridyl) ruthenium(II) (31,32), luciferin-luciferase (33) and permanganate (34) have been developed and coupled to CE for the analysis of various compounds, such as proteins, amino acids, metal ions and so on. Not surprisingly, CL has been considered as a promising detection technique in CE, and has attracted increasing interest (35).…”