Double Coupling Edman Chemistry for High-Sensitivity Automated Protein Sequencing

Abstract

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a promising new method for the analysis of protein sequencing products. It gives 10 zmol (1 zmol = 10(-21) mol) limits of detection (3 sigma) for fluorescein thiohydantoin (FTH) amino acids. We have developed a separation for the (FTH)-amino acid products generated from 18 of the 20 coded amino acids. The extremely low volume requirement associated with CE-LIF makes it incompatible with commercial sequencers. For this reason, we have also been developing a miniaturized sequencer that can be more easily coupled to our detection system. Both the CE-LIF system and the miniaturized sequencer are described.

LC/MS/MS Analysis of N‐Terminal Protein Adducts with Improved Sensitivity: A Comparison of Selected Edman Isothiocyanate Reagents

“…The ionic group and fluorescent properties of the analyte also provides new alternatives such as CE separation and spectroscopic detection, for example, with sensitive fluorescence detection. Ireland et al showed, for example, that they could measure FTH-amino acids with a limit of detection on a low zeptomol (10 −21 ) level per injection by using CE separation with laser induced fluorescence detection [21]. …”

“…Ireland et al [21] showed that amino acids in proteins could be sequenced with FITC as Edman reagent and measured by CE-LIF with outstanding sensitivity. In order to test the same principle for adduct measurements without having access to a CE-LIF system we used CE-DAD to study the separation between FTH-Val and FTH-MeVal and measured the fluorescence properties separately.…”