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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.

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.

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.

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.

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.

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