Prostate-specific antigen (PSA) is the usual biomarker for prostate cancer (PCa). However, its lack of selectivity has lead to the search for new biomarkers. PSA glycosylation seems to depend on the pathophysiological conditions of the individual. Thus, methods to separate PSA isoforms (peaks) to study their role as PCa markers are needed. In this work, CE methods for PSA isoforms separation, based on the use of different dynamic coatings, are developed using UV detection. Three complementary CE methods allowing the separation of 8 or 9 PSA isoforms are selected. The longest method takes only 17 min, while the shortest one separates 9 isoforms in < 8 min. Depending on the isoforms of interest for their use as PCa biomarker, the CE method to be used can be chosen or various of them can be combined. A remarkable aspect of these methods is that the BGEs employed are devoid of compounds with primary amino groups, making the CE methods compatible with fluorescent on-column derivatization through amino residues. As a proof-of-concept, a preliminary result shows that LIF detection of labeled PSA analyzed by one of the three developed methods permits detection of glycoprotein isoforms.
The analysis of glycoprotein isoforms is of high interest in the biomedical field and clinical chemistry. Many studies have demonstrated that some glycoprotein isoforms could serve as biomarkers for several major diseases, such as cancers and vascular diseases, among others. Capillary zone electrophoresis (CZE) is a well‐established technique to separate glycoprotein isoforms, however, it suffers from limited sensitivity when UV‐Vis detection is used. On the other hand, with laser‐induced fluorescence (LIF) detection, derivatization reaction to render the proteins fluorescent can destroy the resolution of the isoforms. In this work, a derivatization procedure through the thiol groups of glycoproteins using either 5‐(iodoacetamide) fluorescein (5‐IAF) or BODIPY iodoacetamide is presented with the model protein of alpha‐1‐acid glycoprotein (AGP). The derivatization process presented enabled high‐resolution analysis of AGP isoforms by CZE‐LIF. The derivatization procedure was successfully applied to label AGP from samples of serum and secretome of artery tissue, enabling the separation of the AGP isoforms by CE‐LIF in natural samples at different concentration levels.
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.
Sample preparation and laser-induced fluorescence detection are two key steps of the analytical methodology to determine by capillary electrophoresis low concentrations of proteins in complex sample matrices. In this chapter the options of performing both steps in different ways are shown by detailing the analysis of the allergen β-lactoglobulin in food products for infants and the analysis of the isoforms of alpha 1-acid glycoprotein, a potential biomarker, in serum and secretome.
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