title: On-capillary fluorescent labeling and capillary electrophoresis laser-induced fluorescence analysis of glycoforms of intact prostate-specific antigen.
abstract: The test used in clinics as prostate cancer (PCa) biomarker, based on the concentration of the glycoprotein prostate-specific antigen (PSA) in serum, leads to an elevated number of false positives. In the search for new PCa biomarkers, analysis of the proportions of different groups of glycoforms of PSA is promising. Peaks of PSA, called isoforms and containing one or several glycoforms of the glycoprotein, can be separated by CE. For those samples in which PSA concentration is very low, a very sensitive detection technique, such as LIF, would be required. However, CE separation of fluorescently labeled isoforms of glycoproteins is challenging. In this work, three different methods of fluorescent derivatization of PSA were assayed with the aim of finding conditions allowing labeling of the glycoprotein compatible with CE resolution of its isoforms. NanoOrange, as a noncovalent label; 5-(iodoacetamide) fluorescein and BODIPY® FL C1 -IA, as covalent tags of thiol groups; and Chromeo™ P503, as a covalent tag of amino groups, were tried. Only the derivatization with the P503 fluorogenic dye led to the resolution by CE-LIF of several isoforms of labeled PSA. Adapting this derivatization method to be performed on-column leads to a reduction in labeling time from 4 h to 45 s. Automation of the whole analysis permitted to carry out fluorescent labeling and CE separation of PSA isoforms in less than 12 min.
authors.affiliation: Institute of Organic Chemistry (CSIC), Madrid, Spain.
authors.forename: Raul
authors.initials: R
authors.lastname: Garrido-Medina
authors.forename: Jose Carlos
authors.initials: JC
authors.lastname: Diez-Masa
authors.forename: Mercedes
authors.initials: M
authors.lastname: de Frutos
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D017430:Prostate-Specific Antigen / Q000032:analysis* / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D006023:Glycoproteins; D013438:Sulfhydryl Compounds; D017430:Prostate-Specific Antigen
keywords: CE-LIF; Fluorescent derivatization; Prostate cancer; Prostate-specific antigen; Protein isoforms