Estimation of protein concentration at high sensitivity using SDS-capillary gel electrophoresis-laser induced fluorescence detection with 3-(2-furoyl)quinoline-2-carboxaldehyde protein labeling

Abstract

3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) is a sensitive fluorogenic dye, used for derivatization of proteins for SDS-CGE with LIF detection (SDS-CGE-LIF) at silver staining sensitivity (ng/mL). FQ labels proteins at primary amines, found at lysines and N-termini, which vary in number and accessibility for different proteins. This work investigates the accuracy of estimation of protein concentration with SDS-CGE-LIF in real biological samples, where a different protein must be used as a standard. Sixteen purified proteins varying in molecular weight, structure, and sequence were labeled with FQ at constant mass concentration applying a commonly used procedure for SDS-CGE-LIF. The fluorescence of these proteins was measured using a spectrofluorometer and found to vary with a RSD of 36%. This compares favorably with other less sensitive methods for estimation of protein concentration such as SDS-CGE-UV and SDS-PAGE-Coomassie and is vastly superior to the equivalently sensitive silver stain. Investigation into the number of labels bound with UHPLC-ESI-QTOF-MS revealed large variations in the labeling efficiency (percentage of labels to the number of labeling sites given by the sequence) for different proteins (from 3 to 30%). This explains the observation that fluorescence per mole of protein was not proportional to the number of lysines in the sequence.

Keywords: 3-(2-Furoyl)quinoline-2-carboxaldehyde; Laser-induced fluorescence detection; Protein quantification; Sodium dodecyl sulfate - capillary gel electrophoresis; Ultrasensitive detection.