Fluorescein-based low-molecular-mass markers of isoelectric point for tracing pH gradient using high sensitivity capillary isoelectric focusing with laser-induced fluorescence detection

Background: Successful implementation of capillary isoelectric focusing (cIEF) in general requires the use of reference points- markers of isoelectric point. The low-molecular-mass fluorescent compounds based on the fluorescein structure, capable of marking distinct isoelectric points (pIs) I s) with good focusing ability, stability, high extinction coefficient, and fluorescence are an alternative to the peptide and protein pI I markers. Results: Four synthetic pathways were used to produce the ampholytic compounds and the structures along with their basic physical and chemical properties are given. Previously published low-molecular-mass pI I markers were used to calibrate the candidate fluorescent compounds using capillary IEF (cIEF) with laser-induced fluorescence (LIF) detection. Linear and point-to-point pI I calibration approach were compared for the pI I evaluation. The results show 21 fluorescent pI I markers (FPIMs). This group of markers covers pH range from 3.10 to 10.21. The usability of the developed FPIMs was further demonstrated by analysis of fluorescein isothiocyanate (FITC) labeled immunoglobin (IgG) standard. Significance and novelty: pI I markers are crucial for tracking the pH gradient and pI I determination of biological ampholytic compounds. This paper presents and characterizes a novel pI I marker set capable of tracing isoelectric points and supporting highly sensitive LIF detection-based applications. A basis of a newly developed cIEF-based fractionation method is described as well.