Monitorization of α1-Acid Glycoprotein Deglycosylation Using SU-8 Microchips Electrophoresis with LIF Detection.

In the last few years, biopharmaceuticals-therapeutic drugs which are generally obtained by using molecular biology techniques-have become a major growing sector in pharmaceutical industry. A large part of these biopharmaceuticals are therapeutic glycoproteins. The production of these drugs and their purification process are implying the development of efficient analytical methods, which allow quick and reliable control of the manufacturing process and ensuring the regulatory compliance about the quality of these drugs. Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is becoming a method of choice in the quality control of these biopharmaceuticals. On the other hand, CGE can be improved if analyses are carried out in microchip format.This chapter reports a detailed microchips gel electrophoresis (MGE) method to separate glycosylated and deglycosylated forms of α1-acid glycoprotein (AGP) labeled with Chromeo P540, using SU-8 microchips and laser induced fluorescence detection. Due to the analogy between AGP and some therapeutic glycoproteins, we have selected AGP as a model system to illustrate the potential of MGE in the analysis of this type of biopharmaceutical compounds.

Capillary Electrophoresis Analysis of Prostate-Specific Antigen (PSA).

The Capillary Electrophoresis (CE) profile of isoforms (peaks) of a glycoprotein can be useful to show alterations in its posttranslational modifications (PTMs) linked to diseases. These changes can modify the electrophoretic mobility of these isoforms in a minor extent and, therefore, very reproducible CE methods are needed to detect them. In this chapter, a method for the analysis of prostate-specific antigen (PSA) by Capillary Zone Electrophoresis (CZE) with UV detection is detailed. High reproducibility in the separation of a large number of PSA isoforms is achieved by performing capillary conditioning in acid media and by using a background electrolyte (BGE) at pH 8.0 formulated with decamethonium bromide and urea.

Comparative analysis of prostate-specific antigen by two-dimensional gel electrophoresis and capillary electrophoresis.

Serum levels of Prostate-Specific Antigen (PSA) are not fully specific for prostate cancer (PCa) diagnosis and several efforts are focused on searching to improve PCa markers through the study of PSA subforms that could be cancer associated. We have previously reported by 2DE a decrease in the sialic acid content of PSA from PCa compared to benign prostatic hyperplasia patients based on the different proportion of the PSA spots. However, faster and more quantitative techniques, easier to automate than 2DE, are desirable. In this study, we examined the potential of CE for resolving PSA subforms in different samples and compared the results with those obtained by 2DE. We first fractionated by OFFGEL the subforms of PSA from seminal plasma according to their pIs and analyzed each separated fraction by 2DE and CE. We also analyzed PSA and high pI PSA, both from seminal plasma, and PSA from urine of a PCa patient. These samples with different PSA spots proportions by 2DE, due to different posttranslational modifications, also presented different CE profiles. This study shows that CE is a useful and complementary technique to 2DE for analyzing samples with different PSA subforms, which is of high clinical interest.

Impact of capillary conditioning and background electrolyte composition on capillary electrophoresis analysis of prostate specific antigen isoforms.

Glycoproteins expressed in the human body can experience modifications as result of pathological situations. Detection of those changes can be useful as disease biomarkers. As a result of these modifications, size and/or electrical charge of the glycoprotein can be altered. Migration in capillary zone electrophoresis (CZE) is governed by the size to charge ratio of the analyte and therefore this separation technique can be used to monitor those modifications. At its turn, the alteration of the electrophoretical pattern of a given glycoprotein could be used as disease biomarker. To this aim, high repeatability for separation of a large number of peaks for a given glycoprotein is desirable. For prostate cancer, new markers are needed to decrease the high number of false positive results provided by the biomarkers currently used in clinics. In this sense, CZE methods for analysis of the several prostate specific antigen (PSA) peaks which this glycoprotein exhibit, called isoforms and containing one or more glycoforms, could be useful to study the PSA pattern as prostate cancer marker. In this study two complementary strategies to achieve both lot-to-lot capillary repeatability and high resolution of a large number of PSA isoforms are developed. Better performance and precision have been obtained for capillaries conditioned with HCl than for those conditioned with NaOH. Optimization of the background electrolyte (BGE) pH value to 8.0 and inclusion of 3M urea on its composition were the two factors of highest impact for enhancing resolution of the highest number of PSA peaks. Under the optimized conditions for capillary conditioning and BGE pH and composition, long-term resolution of 10 isoforms of PSA was achieved. Inter-day (n=3) %RSD was 0.55 for the ratio tm/tEOF, 1.15 for µeff, and 5.02 for % Acorr of the PSA peaks.

Immunoaffinity chromatographic isolation of prostate-specific antigen from seminal plasma for capillary electrophoresis analysis of its isoforms.

Prostate-specific antigen (PSA) concentration in serum has been the biomarker employed for prostate cancer diagnosis in the last two decades. However, new more specific biomarkers allowing a better differentiation of cancer from non-malignant prostate diseases are necessary. Glycosylation of PSA gives rise to different forms of the protein which can be separated into several isoforms by analytical techniques, such as CE. Because PSA glycosylation is influenced by pathological conditions, the CE pattern of PSA isoforms could be different in prostate cancer than in non-malignant prostate diseases. To study this CE pattern of PSA, prior purification of the protein from the biological fluid is mandatory. In this study an immunoaffinity chromatography method which allows PSA purification without altering the CE pattern is developed. An in-house prepared column produced with commercial anti-PSA antibodies is employed. The use of 1 M propionic acid as elution agent provides higher than 40% recovery of high purity PSA. CE analysis of PSA immunopurified from seminal plasma of a healthy individual shows the same 8 peaks as the commercially available PSA standard. Sample preparation only requires dilution with phosphate buffered saline prior to immunoaffinity purification. High repeatability for the sample preparation step was achieved (RSD% for percentage of corrected peak area in the range 0.6-5.3 for CE analysis of three independently purified seminal plasma aliquots compared to range 0.8-4.9 for a given aliquot analyzed three times by CE). IAC of five microliters seminal plasma provided enough PSA to achieve signal/noise ratio larger than 5 for the smallest CE isoforms.

Development of an SDS-gel electrophoresis method on SU-8 microchips for protein separation with LIF detection: Application to the analysis of whey proteins.

This work describes the development of an SDS-gel electrophoresis method for the analysis of major whey proteins (α-lactalbumin, ß-lactoglobulin, and BSA) carried out in SU-8 microchips. The method uses a low-viscosity solution of dextran as a sieving polymer. A commercial coating agent (EOTrol LN) was added to the separation buffer to control the EOF of the chips. The potential of this coating agent to prevent protein adsorption on the walls of the SU-8 channels was also evaluated. Additionally, the fluorescence background of the SU-8 material was studied to improve the sensitivity of the method. By selecting an excitation wavelength of 532 nm at which the background fluorescence remains low and by replacing the mercury arc lamp by a laser in the detection system, an LOD in the nanomolar range was achieved for proteins derivatized with the fluorogenic reagent Chromeo P540. Finally, the method was applied to the analysis of milk samples, demonstrating the potential of SU-8 microchips for the analysis of proteins in complex food samples.

On-capillary fluorescent labeling and capillary electrophoresis laser-induced fluorescence analysis of glycoforms of intact prostate-specific antigen.

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.

On-line immunoaffinity capillary electrophoresis based on magnetic beads for the determination of alpha-1 acid glycoprotein isoforms profile to facilitate its use as biomarker.

An immunoaffinity purification method coupled on-line to capillary electrophoresis (IACE) which allows the determination of several isoforms of intact alpha-1 acid glycoprotein (AGP) in serum samples using UV detection is developed. The immunoaffinity step is based on anti-AGP antibodies (Abs) covalently bound to magnetic beads (MBs) which are captured at the inlet end of the capillary using permanent magnets placed inside the cartridge of the CE instrument. The on-line method includes injection of the MBs with the Ab bound (MBs-Ab) and their trapping by the magnets at the entrance of the separation column, injection of serum sample and capture of AGP by the Abs, release of captured AGP, focus of desorbed protein, separation of AGP isoforms, and removal of MBs-Ab. The optimization of the different factors involved in each step allowed purification, separation and detection of AGP isoforms in a single electrophoretic analysis in about 1 h. Automation, sample and reagents consumption as well as analysis time was improved compared to off-line alternatives which use purification of AGP in an immunochromatographic column and CE separation of AGP isoforms in two independent operations. The analytical methodology developed allows the separation of 10 AGP isoforms in serum samples from a healthy donor. For a serum sample, precision (expressed as relative standard deviation) in terms of corrected area percentage was better than 0.5% for each peak accounting for more than 10% of total AGP and it was better than 4.0% in terms of relative migration time of each AGP isoform considering the whole process.

Capillary electrophoresis with laser-induced fluorescence detection of proteins from two types of complex sample matrices: food and biological fluids.

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 ß-lactoglobulin in food products for infants and the analysis of the isoforms of alpha 1-acid glycoprotein, a potential biomarker, in serum and secretome.

Protein fingerprinting of Staphylococcus aureus by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection.

This chapter describes a complete procedure for obtaining protein fingerprints of microorganisms using capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). Staphylococcus aureus, a human pathogen responsible of frequent and resistant infections, is used as model microorganism to show the feasibility of this procedure. Bacteria are grown in different culture media or submitted to temperature or nitrosative stress conditions. After the growth of the bacteria, the protein extracts are obtained by cell lysis using sonication. The water-soluble fraction of these lysates is derivatized on-capillary with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. The fluorescent products are analyzed by CE and detected by LIF. Practical advices for the interpretation of the electropherograms are given. To do so, the variations of the protein fingerprints of the bacteria with the culture conditions, such as growth medium, or the stressing conditions, such as heat shock or nitrosative stress, are used as example.

Analysis of alpha-1-acid glycoprotein isoforms using CE-LIF with fluorescent thiol derivatization.

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.

High resolution separation methods for the determination of intact human erythropoiesis stimulating agents. A review.

Human erythropoietin (hEPO), a hormone involved in the formation of red blood cells, is a 30 kDa glycoprotein with a high carbohydrate content. The production of recombinant hEPO has made possible its widespread therapeutic use and its banned use in competition sports. Methods to analyze EPO and other erythropoiesis stimulating agents (ESAs) are necessary for the characterization and quality control of these biopharmaceuticals and also for doping control. In this paper, high resolution separation methods, namely high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), with special attention to CE-coupled mass spectrometry, are reviewed. The usefulness of these techniques when applied in different modes to separate the glycoprotein isoforms, aggregates or excipients are detailed. In addition, sample preparation methods that have been applied to ESA samples for subsequent determination by HPLC or CE, as well as the potential compatibility of other preparation methods, are discussed. Applications of the HPLC and CE methods regarding regulatory considerations for biopharmaceuticals analysis, with emphasis on biosimilars, and doping control are also included. Finally, limitations of the present methods and their possible solutions are considered.

CE methods for analysis of isoforms of prostate-specific antigen compatible with online derivatization for LIF detection.

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.

Study of the capillary electrophoresis profile of intact α-1-acid glycoprotein isoforms as a biomarker of atherothrombosis.

α-1-Acid glycoprotein (AGP) is a serum glycoprotein that presents several isoforms. Changes in the isoforms of AGP have been related to different pathological states including cardiovascular diseases (CVDs) such as acute myocardial infarction. However, to our knowledge, the role of variations of AGP isoforms as a potential biomarker of atherothrombosis has not been addressed. In this work, a preliminary study about differences in the capillary zone electrophoresis (CZE) profile of intact (non-hydrolyzed) AGP isoforms between healthy individuals and patients with atherothrombosis, specifically abdominal aortic aneurysm (AAA) and carotid atherosclerosis (CTA), has been performed. Biological samples (plasmas and sera) were analyzed by CZE after immunoaffinity chromatography purification. Up to 13 peaks corresponding to groups of isoforms of intact AGP from plasma samples were detected by CZE-UV. Electrophoretic profiles were aligned, peaks assigned, and linear discriminant analysis (LDA) of percentage of the corrected areas of AGP peaks was employed to discriminate and classify the CZE profiles of AGP samples. LDA enabled to accomplish 92.9% of correct classification of the AGP samples when the three groups of samples were considered. Besides, the LDA model showed high predictive power in the groups healthy vs. sick, healthy vs. AAA, and healthy vs. CTA. The described method was a successful approach to study the potential of AGP isoforms profile as a biomarker of atherothrombosis. To the best of our knowledge this has been the first time that a possible role of the CZE profile of intact AGP isoforms as a biomarker of vascular diseases has been demonstrated.

Evaluation of the effect of the immunopurification-based procedures on the CZE-UV and CZE-ESI-TOF-MS determination of isoforms of intact alpha-1-acid glycoprotein from human serum.

Differences in alpha-1-acid glycoprotein (AGP) peptidic and glycan moieties originate several isoforms, whose modifications have been related to different pathophysiological situations. Differences in the isoforms of AGP existing in serum of individuals suffering from different diseases compared to healthy ones could be potentially used as biomarkers. CZE has been proven to be a useful technique for the analysis of glycoprotein isoforms. However, direct CZE analysis of AGP isoforms in serum samples needs efficient purification methods that allow the protein analysis. In this work two new and fast methods to purify AGP from human serum are evaluated in regard to their effect on the determination of isoforms of the intact glycoprotein by CZE-UV and by a developed CZE-ESI-TOF-MS method. Both preparation methods, which differ in the pre-treatment of the sample prior to an anti-AGP immunochromatographic step are shown to be adequate to analyze isoforms of intact AGP. Comparison of both purification methods by CZE-UV and CZE-ESI-TOF-MS indicates that serum AGP purified without acidic precipitation as pre-treatment is more adequate due to AGP higher yield, which leads to better CZE-Mass spectra. Both CZE methods show no indication that acidic precipitation influences the glycosylation (including sialylation) of AGP.

Development of a fast and simple immunochromatographic method to purify alpha 1-acid glycoprotein from serum for analysis of its isoforms by capillary electrophoresis.

Alpha 1-acid glycoprotein (AGP) is a very heterogeneous glycoprotein presenting several isoforms due to variations in its polypeptidic and glycosidic moieties. Differences in AGP isoforms between healthy and diseased individuals have been related to different pathological situations such as cancer or cardiovascular diseases, among others. Capillary electrophoresis study of the role of AGP isoforms as biomarkers requires prior purification of AGP from biological samples. Current AGP purification methods are time- and labour-consuming, and generally they have not been proven to be compatible with capillary electrophoresis analysis. In this work, different methods for AGP purification from human serum are developed and compared. The applicability of acidic precipitation and immunoaffinity chromatographic methods for AGP purification are studied. Two different immunoaffinity approaches are employed; in the first one, interferents present in the AGP sample are captured and removed, and in the second one, AGP is retained in a house-made anti-AGP column, being in this way isolated from the rest of interferents of the sample. Best results in AGP purification from human serum to be analyzed by capillary zone electrophoresis (CZE) were obtained when acidic purification was combined with immunoaffinity chromatography (IAC) employing the house-made anti-AGP column. The method was shown not to alter the proportion of AGP peaks due to isoforms existing in AGP samples. The applicability of this fast and easy purification method developed for analyzing by CZE isoforms of AGP from natural serum samples by CZE is demonstrated.

Development of an optimized ELISA and a sample preparation method for the detection of beta-lactoglobulin traces in baby foods.

Dairy products can induce allergic reactions even when present at very low levels, such as levels found in involuntary contamination during food manufacturing. beta-Lactoglobulin (betaLG) is the main allergen in cow's milk. The objective of this work was to develop a sensitive method for betaLG detection in baby foods through the optimization of an innovative sample preparation method. Three types of baby foods deliberately contaminated with dairy products or dairy desserts were sterilized to simulate the potential contamination occurring during manufacturing and then used as samples. Different sample preparation methods were compared. The best results were provided by an extraction solution containing beta-mercaptoethanol, guanidine hydrochloride, and a saline solution. An ELISA method was optimized for the detection of betaLG (LOD = 9.7 x 10(-13) M). The developed method allowed detection of even 1 part of dairy product in 100,000 parts of baby food for some of the analyzed foods.

Protein fingerprinting of Staphylococcus species by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection.

Capillary electrophoresis (CE) coupled with laser-induced fluorescence detection (LIF) has allowed to obtain protein fingerprints, which have demonstrated to be useful in microorganisms characterization. In this work, protein fingerprints of two species of Staphylococcus grown in different culture media and submitted to temperature and nitrosative stress were studied by CE-LIF. After the growth of the bacteria, protein extracts were obtained by cell lysis using sonication. The water-soluble fraction of these lysates was derivatized on-capillary with a fluorogenic dye, 3-(2-furoyl)quinoline-2-carboxaldehyde. The fluorescent products were analyzed by CE using phosphate buffer containing submicellar concentrations of sodium pentanesulfate and detected by LIF. Different protein fingerprints were obtained depending on the bacterial species studied, indicating the usefulness of this method for the identification of different species of the same bacterial genus. It was also demonstrated that the CE protein fingerprints were dependent on the culture conditions, such as growth medium, or on stressing conditions, such as heat shock or nitrosative stress.

A new sample preparation method compatible with capillary electrophoresis and laser-induced fluorescence for improving detection of low levels of beta-lactoglobulin in infant foods.

Beta-lactoglobulin (betaLG) is the main allergenic protein in cow's milk and can cause allergy even when present at very low concentration. The aim of this work is to develop an innovative sample preparation method fully compatible with capillary electrophoresis and laser-induced fluorescence detection for improving the sensitivity when analyzing betaLG. Different types of baby food were on purpose contaminated with diverse dairy desserts and submitted to thermal treatment to simulate potential contamination at production. Sample preparation prior to CE analysis was performed by the classical extraction method and by the innovative one, and the results were compared. Analysis was performed by capillary electrophoresis with laser-induced fluorescence detection. The innovative method permitted to detect contaminations as low as 1 part of yoghurt in 10,000 parts of baby food.

CIEF and MALDI-TOF-MS methods for analyzing forms of the glycoprotein VEGF 165.

The vascular endothelial growth factor (VEGF) is involved in different sicknesses (cardiovascular diseases, cancer, and other). Out of the many components of the VEGF family, the A splice variant with 165 amino acids (VEGF(165)) is the main component. In spite of the potential as biomarker that this protein has, information about its physico-chemical characteristics is scarce. In this study CIEF and MALDI-TOF-MS methods for intact recombinant human VEGF(165) are developed and applied to analyze this glycoprotein expressed in glycosylating (Sf 21 insect cells) and non-glycosylating (Escherichia coli) systems. Different parameters influencing the CIEF separation were studied. The developed CIEF method allowed for the separation of up to seven peaks in the VEGF(165) expressed in insect cells and up to three in VEGF(165) expressed in E. coli. The use of the presented method permits the estimation of the apparent pI of the different forms of VEGF(165) expressed in insect cells to be in a range of 6.8-8.2. The three peaks with intermediate pI values are observed in the protein expressed in both systems, insect cells and E. coli. The MALDI-TOF-MS method enabled to a rapid partial characterization of VEGF(165) based on its MS fingerprint. MALDI-MS analysis of VEGF(165) expressed in insect cells shows the presence of, at least, four forms or groups of forms of VEGF(165) as a result of the different PTMs of the protein. According to the MALDI-MS analysis, VEGF(165) expressed in E. coli was produced as a very homogeneous protein, although the results suggest the existence of some PTMs in the protein. The patterns of VEGF(165) of both origins obtained by CIEF and MALDI-MS indicate the possibility of using these analytical methods to compare samples from people with different pathophysiological conditions. This work is thus a starting point to make possible the study of the role of the various forms of VEGF(165) as biomarkers. Finally, to the best of our knowledge, this is the first time that intact VEGF(165) has been analyzed by CIEF and MALDI-TOF-MS.