Institute of Organic Chemistry CSIC

San Juan de la Rambla, Spain

Institute of Organic Chemistry CSIC

San Juan de la Rambla, Spain
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Ongay S.,Institute Of Organic Chemistry Csic | Neususs C.,Aalen University of Applied Sciences
Analytical and Bioanalytical Chemistry | Year: 2010

Human AGP is an acidic glycoprotein mainly produced by liver that presents a high degree of heterogeneity. It can present different amino acid sequences and has five N-glycosylation sites leading to a wide range of different protein isoforms. AGP structure and composition has been widely studied due to its drug-binding behavior and relation with disease. However, so far, the characterization has been performed only on protein fragments, i.e., the peptide or glycan level. Here, the analysis of intact human AGP purified from human serum is performed by capillary electrophoresis-time-of-flight mass spectrometry. In this way, it is possible to characterize more than 150 human AGP isoforms, differing both in the amino acid sequence and in the glycosylation. The detected masses could be attributed unequivocally to an overall composition based on the combination of the analysis of the released glycans and the characterization of the deglycosylated protein. Different AGP samples purified from human serum were characterized and compared. High inter-individual variability among AGP isoforms expression was observed. The presented method enables for the first time clinical studies based on detailed isoform distribution of intact glycoproteins. © 2010 Springer-Verlag.


Ongay S.,Institute of Organic Chemistry CSIC | Martin-Alvarez P.J.,Institute of Industrial Fermentations CSIC | Neusu C.,Aalen University of Applied Sciences | de Frutos M.,Institute of Organic Chemistry CSIC
Electrophoresis | Year: 2010

α-1-Acid glycoprotein (AGP) is a highly heterogeneous protein that presents a vast number of isoforms (molecules of the protein differing in its peptidic and/or glycosidic moieties). In recent years, several authors have studied the potential use of AGP as a cancer biomarker. These studies focus on the correlation of different features of AGP structure (i.e. fucosylation, antennarity) with cancer or on the total protein blood concentration. In this study, the potential of CZE-UV and CZE-ESI-MS analysis of intact AGP isoforms to study the correlation of this protein with bladder cancer is shown. Samples from 16 individuals (eight healthy, eight bladder cancer) were analyzed and characterized in great detail including data on intact protein isoforms and on released glycans. The analytical data were evaluated employing different statistical techniques (ANOVA; principal component analysis, linear discriminant analysis; and partial least squares-discriminant analysis). Statistical differences between the two groups of study were observed. The best results were obtained by linear discriminant analysis of the CZEESI-MS data for intact AGP isoforms (93.75% of correct classification). Due to MS characterization, it can be observed that differences between the samples are mainly due to higher abundance of AGP isoforms containing tri- and tetra-antennary fucosylated oligosaccharides in cancer patients. The results show the great potential of CE-MS in combination with advanced data processing for the use of intact protein isoforms as disease biomarkers. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Ongay S.,Institute Of Organic Chemistry Csic | Neususs C.,Aalen University of Applied Sciences | Vaas S.,Aalen University of Applied Sciences | Diez-Masa J.C.,Institute Of Organic Chemistry Csic | De Frutos M.,Institute Of Organic Chemistry Csic
Electrophoresis | Year: 2010

Differences in α-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. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.


Girard M.,Biologics | Puerta A.,Institute of Organic Chemistry CSIC | Diez-Masa J.C.,Institute of Organic Chemistry CSIC | de Frutos M.,Institute of Organic Chemistry CSIC
Analytica Chimica Acta | Year: 2012

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. © 2011 Elsevier B.V.


Pelaez-Lorenzo C.,Institute of Organic Chemistry CSIC | Diez-Masa J.C.,Institute of Organic Chemistry CSIC | Vasallo I.,Hero Espana S.A. | De Frutos M.,Institute of Organic Chemistry CSIC
Journal of Agricultural and Food Chemistry | Year: 2010

Dairy products can induce allergic reactions even when present at very low levels, such as levels found in involuntary contamination during food manufacturing. β-Lactoglobulin (βLG) is the main allergen in cow's milk. The objective of this work was to develop a sensitive method for βLG 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 β-mercaptoethanol, guanidine hydrochloride, and a saline solution. An ELISA method was optimized for the detection of βLG (LOD = 9.7 × 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. © 2010 American Chemical Society.


Ozohanics O.,Hungarian Academy of Sciences | Turiak L.,Hungarian Academy of Sciences | Puerta A.,Institute of Organic Chemistry CSIC | Vekey K.,Hungarian Academy of Sciences | Drahos L.,Hungarian Academy of Sciences
Journal of Chromatography A | Year: 2012

Analysis of protein glycosylation is a major challenge in biochemistry, here we present a nano-UHPLC-MS(MS) based methodology, which is suitable to determine site-specific N-glycosylation patterns. A few pmol glycoprotein is sufficient to determine glycosylation patterns (which opens the way for biomedical applications) and requires at least two separate chromatographic runs. One is using tandem mass spectrometry (for structure identification); the other single stage MS mode (for semi-quantitation). Analysis relies heavily on data processing. The previously developed GlycoMiner algorithm and software was used to identify glycopeptides in MS/MS spectra. We have developed a new algorithm and software (GlycoPattern), which evaluates single stage mass spectra, both in terms of glycopeptide identification (for minor glycoforms) and semi-quantitation. Identification of glycopeptide structures based on MS/MS analysis has a false positive rate of 1%. Minor glycoforms (when sensitivity is insufficient to obtain an MS/MS spectrum) can be identified in single stage MS using GlycoPattern; but in such a case the false positive rate is increased to 5%. Glycosylation is studied at the glycopeptide level (i.e. following proteolytic digestion). This way the sugar chains can be unequivocally assigned to a given glycosylation site (site-specific glycosylation pattern). Glycopeptide analysis has the further advantage that protein-specific glycosylation patterns can be identified in complex mixtures and not only in purified samples. This opens the way for medium high throughput analysis of glycosylation. Specific examples of site-specific glycosylation patterns of alpha-1-acid glycoprotein, haptoglobin and on a therapeutic monoclonal antibody, Infliximab are also discussed. © 2012 Elsevier B.V.


Garrido-Medina R.,Institute of Organic Chemistry CSIC | Diez-Masa J.C.,Institute of Organic Chemistry CSIC | De Frutos M.,Institute of Organic Chemistry CSIC
Electrophoresis | Year: 2013

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. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Del Mar Barrios-Romero M.,Institute of Organic Chemistry CSIC | Crevillen A.G.,Institute of Organic Chemistry CSIC | Diez-Masa J.C.,Institute of Organic Chemistry CSIC
Journal of Separation Science | Year: 2013

This work describes the development of an SDS-gel electrophoresis method for the analysis of major whey proteins (α-lactalbumin, β-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. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Morales-Cid G.,Institute of Organic Chemistry CSIC | Diez-Masa J.C.,Institute of Organic Chemistry CSIC | De Frutos M.,Institute of Organic Chemistry CSIC
Analytica Chimica Acta | Year: 2013

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. © 2013 Elsevier B.V.


Garrido-Medina R.,Institute of Organic Chemistry CSIC | Diez-Masa J.C.,Institute of Organic Chemistry CSIC | De Frutos M.,Institute of Organic Chemistry CSIC
Electrophoresis | Year: 2011

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 17min, while the shortest one separates 9 isoforms in < 8min. 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. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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