title: 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.
authors: Institute of Life Technologies, University of Applied Sciences Western Switzerland (HES-SO Valais/Wallis), Sion, Switzerland.
authors: Miriam S
authors: MS
authors: Arrell
authors: Institute of Life Technologies, University of Applied Sciences Western Switzerland (HES-SO Valais/Wallis), Sion, Switzerland.
authors: Franka
authors: F
authors: Kálmán
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry*; D005663:Furans / Q000737:chemistry*; D016014:Linear Models; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D011804:Quinolines / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D005663:Furans; D011506:Proteins; D011804:Quinolines; C063756:3-(2-furoyl)quinoline-2-carbaldehyde; D012967:Sodium Dodecyl Sulfate
keywords: 3-(2-Furoyl)quinoline-2-carboxaldehyde; Laser-induced fluorescence detection; Protein quantification; Sodium dodecyl sulfate - capillary gel electrophoresis; Ultrasensitive detection