Publications by authors named "Andras Guttman"

179 Publications

Rapid capillary gel electrophoresis analysis of human milk oligosaccharides for food additive manufacturing in-process control.

Anal Bioanal Chem 2021 Mar 8;413(6):1595-1603. Epub 2021 Feb 8.

Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, Doctoral School of Molecular Medicine, University of Debrecen, Debrecen, 4032, Hungary.

Industrial production of human milk oligosaccharides (HMOs) represents a recently growing interest since they serve as key ingredients in baby formulas and are also utilized as dietary supplements for all age groups. Despite their short oligosaccharide chain lengths, HMO analysis is challenging due to extensive positional and linkage variations. Capillary gel electrophoresis primarily separates analyte molecules based on their hydrodynamic volume to charge ratios, thus, offers excellent resolution for most of such otherwise difficult-to-separate isomers. In this work, two commercially available gel compositions were evaluated on the analysis of a mixture of ten synthetic HMOs. The relevant respective separation matrices were then applied to selected analytical in-process control examples. The conventionally used carbohydrate separation matrix was applied for the in-process analysis of bacteria-mediated production of 3-fucosyllactose, lacto-N-tetraose, and lacto-N-neotetraose. The other example showed the suitability of the method for the in vivo in-process control of a shake flask and fermentation approach of 2'-fucosyllactose production. In this latter instance, borate complexation was utilized to efficiently separate the 2'- and 3-fucosylated lactose positional isomers. In all instances, the analysis of the HMOs of interest required only a couple of minutes with high resolution and excellent migration time and peak area reproducibility (average RSD 0.26% and 3.56%, respectively), features representing high importance in food additive manufacturing in-process control.
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http://dx.doi.org/10.1007/s00216-020-03119-0DOI Listing
March 2021

Effect of the Monomer Cross-Linker Ratio on the Separation Selectivity of Monoclonal Antibody Subunits in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis.

Anal Chem 2021 Feb 5;93(7):3535-3541. Epub 2021 Feb 5.

Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, Doctoral School of Molecular Medicine, University of Debrecen, 98 Nagyerdei krt, Debrecen H-4032, Hungary.

With the increasing interest in the biopharmaceutical industry toward novel and innovative protein therapeutics, improved separation techniques are important, especially for the analysis of highly glycosylated candidates. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran is one of the most frequently used methods to analyze biotherapeutic proteins in process control as well as in release and stability testing. In this work, the effect of the monomer (dextran) and cross-linker (borate) ratio was studied in SDS-CGE analysis of a therapeutic monoclonal antibody test item in its reduced and intact forms. A retention model was developed for better understanding of the separation selectivity between the non-glycosylated and glycosylated heavy chain fragments, exploiting the interaction between the dextran-borate adducts and the glycan moiety of the therapeutic antibody. The monomer cross-linker ratio played a significant role in the overall analysis times and affected the separation selectivity between the non-glycosylated and regular (glycosylated) heavy chain fragments; however, it had no effect on the separation of the regular and non-glycosylated intact forms of the monoclonal antibody. Introduction of three-dimensional selectivity plots offered an easy separation optimization option for the separation problem in hand.
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http://dx.doi.org/10.1021/acs.analchem.0c04927DOI Listing
February 2021

Recent advances in the analysis of human milk oligosaccharides by liquid phase separation methods.

J Chromatogr B Analyt Technol Biomed Life Sci 2021 Jan 16;1162:122497. Epub 2020 Dec 16.

Translational Glycomics Research Center, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Hungary; Horváth Csaba Memorial Laboratory for Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary. Electronic address:

Human milk is a complex, dynamically changing biological fluid, which contains a large amount of non-conjugated carbohydrates, referred to as human milk oligosaccharides (HMOs). These HMOs are very important for the infants as they play important roles in the formation of the gut microbiome, the immune system and support brain development. HMOs show highly complex structural diversity due to numerous linkage possibilities of the building monosaccharides. In order to elucidate their structure-function relationship and to develop more effective infant formulas, cutting-edge analytical technologies are in great demand. In this paper, we review the current strategies for HMO analysis based on liquid phase separation methods. High performance liquid chromatography, capillary electrophoresis and their hyphenation with mass spectrometry are critically reviewed, emphasizing their advantages and disadvantages from practical point of views. Recent advances of the methods are categorized according to their application fields.
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http://dx.doi.org/10.1016/j.jchromb.2020.122497DOI Listing
January 2021

Evaluation of Possible Processing Time Effects on the Global N-Glycosylation Profile of Human Blood Samples.

Curr Mol Med 2020 ;20(10):840-846

Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Hungary.

The utilization of N-glycan profiling recently gained high importance in fundamental biomedical and applied clinical research. However, for the time being, no glycan biomarker has been approved for clinical diagnosis by the regulatory agencies due to the lack of verifications on large patient cohorts and suitable analytical technologies. In this paper, the effect of human blood sample handling was studied prior to N-glycosylation profiling by capillary electrophoresis, coupled with high sensitivity fluorescence detection. Special attention was paid to the preservation of sialylated structures because of their important clinical - biological relevance. Our results suggested that it is adequate to refrigerate and store the collected total blood samples prior to analysis to obtain unbiased results. Furthermore, we report on the good practice of serum sample handling in order to prevent decomposition of the sialylated structures. Our findings may promote procedure standardization and easier clinical translation of diagnostic N-glycosylation profiling in molecular medicinal applications.
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http://dx.doi.org/10.2174/1566524020666201230094722DOI Listing
January 2020

Machine Learning Based Analysis of Human Serum glycome Alterations to Follow up Lung Tumor Surgery.

Cancers (Basel) 2020 Dec 9;12(12). Epub 2020 Dec 9.

Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Doctoral School of Molecular Medicine, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary.

The human serum glycome is a valuable source of biomarkers for malignant diseases, already utilized in multiple studies. In this paper, the glycosylation changes in human serum proteins were analyzed after surgical lung tumor resection. Seventeen lung cancer patients were involved in this study and the glycosylation pattern of their serum samples was analyzed before and after the surgery using capillary electrophoresis separation with laser-induced fluorescent detection. The relative peak areas of 21 glycans were evaluated from the acquired electropherograms using machine learning-based data analysis. Individual glycans as well as their subclasses were taken into account during the course of evaluation. For the data analysis, both discrete (e.g., smoker or not) and continuous (e.g., age of the patient) clinical parameters were compared against the alterations in these 21 -linked carbohydrate structures. The classification tree analysis resulted in a panel of glycans, which could be used to follow up on the effects of lung tumor surgical resection.
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http://dx.doi.org/10.3390/cancers12123700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764602PMC
December 2020

Separation based characterization methods for the N-glycosylation analysis of prostate-specific antigen.

J Pharm Biomed Anal 2021 Feb 25;194:113797. Epub 2020 Nov 25.

Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, Faculty of Engineering, University of Pannonia, Veszprem, Hungary; Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Centre for Molecular Medicine, Doctoral School of Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary. Electronic address:

Prostate cancer has the highest malignancy rate diagnosed in men worldwide. Albeit, the gold standard serum prostate-specific antigen (PSA) assays reduced the mortality rate of the disease, the number of false positive diagnoses steeply increased. Therefore, there is an urgent need for complementary biomarkers to enhance the specificity and selectivity of current diagnostic methods. Information about PSA glycosylation can help to fulfill this gap as alterations of its carbohydrate moieties due to cancerous transformation may represent additional markers to distinguish malignant from benign tumors. However, development of suitable methods and instrumentations to investigate the N-glycosylation profile of PSA represents a challenge. In this paper, we critically review the current bioanalytical trends and strategies in the field of PSA glycobiomarker research focusing on separation based characterization methods.
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http://dx.doi.org/10.1016/j.jpba.2020.113797DOI Listing
February 2021

N-glycan Analysis in Molecular Medicine: Innovator and Biosimilar Protein Therapeutics.

Curr Mol Med 2020 ;20(10):828-839

Horvath Csaba Memorial Institute of Bioanalytical Research, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Hungary.

The market segment of new biological drugs (monoclonal antibodies, fusion proteins, antibody-drug conjugates, and new modality protein therapeutics) is rapidly growing, especially after the patent expiration of the original biologics, initiating the emergence of biosimilars. N-glycosylation of therapeutic proteins has high importance on their stability, safety, immunogenicity, efficacy, and serum half-life. Therefore, Nglycosylation is considered to be one of the critical quality attributes. Consequently, it should be rigorously monitored during the development, manufacturing, and release of glycoprotein biologicals. In this review, first, the regulatory considerations for biosimilars are shortly summarized, followed by conferring the analytical techniques needed for monitoring and characterization of the N-glycosylation of biological drugs. Particular respect is paid to liquid phase separation techniques with high sensitivity and highresolution detection methods, including laser-induced fluorescence and mass spectrometry.
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http://dx.doi.org/10.2174/1566524020999201203212352DOI Listing
January 2020

Determination of complex type free, non-conjugated oligosaccharide glucose unit values in tomato xylem sap for early detection of nutrient deficiency.

Electrophoresis 2021 Feb 22;42(3):200-205. Epub 2020 Nov 22.

Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, Doctoral School of Molecular Medicine, University of Debrecen, Hungary.

Although knowledge on glycan biosynthesis and processing is continuously maturing, there are still a limited number of studies that examine biological functions of N-glycan structures in plants, which remain virtually unknown. Here, the statistical correlation between nutrient (nitrogen) deficiency symptoms of crops and changes in 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled complex type free oligosaccharides is reported. While deficiency symptoms are predicted by multispectral images and Kjeldahl digestion, APTS-labeled complex type free oligosaccharides are identified by their glucose unit (GU) values in tomato xylem sap, using capillary electrophoresis with laser induced fluorescence detection (CE-LIF). Given the limited number of structures obtained from plants, archived in the literature, in the future, it is intended to create an open access database of promising indicators, namely, glycan structures that are presumably responsible for the nutrient deficiency caused stress in plants (http://glycoplants.org).
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http://dx.doi.org/10.1002/elps.202000254DOI Listing
February 2021

Ultrafast high-resolution analysis of human milk oligosaccharides by multicapillary gel electrophoresis.

Food Chem 2021 Mar 28;341(Pt 2):128200. Epub 2020 Sep 28.

Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, Doctoral School of Molecular Medicine, University of Debrecen, Hungary; Translation Glycomics Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Hungary. Electronic address:

There is recently growing interest towards synthesized human milk oligosaccharides (HMOs) as baby formula additives, and interestingly also as dietary supplements for adults. Currently quite a few manufacturers synthesize HMOs, however, their analysis is challenging, both in resolution and speed. In this paper an ultrafast high-resolution method is introduced for the separation of HMOs by multicapillary gel electrophoresis. Two gel compositions were evaluated with complementary resolving power. One was a conventionally used industrial standard carbohydrate separation matrix, resolving oligosaccharides according to their charge to hydrodynamic volume ratios. The other one was a borate-buffered dextran gel, which utilized the secondary equilibrium of the borate-vicinal diol complexation to enhance resolution. Considering the rapid analysis time and multiplexing (12-channel system), a 96 well sample plate can be analyzed in less than 80 min with the conventional type carbohydrate separation matrix and in less than one hour with the borate-buffered dextran gel. Exploiting the one fluorophore per molecule labeling stoichiometry, the limit of detection (S/N > 3) and limit of quantitation (S/N > 10) were determined as 0.025 and 0.100 mg/mL, respectively, with good linearity. Based on the calibration plot, the quantities of several low concentration HMOs were determined from a human milk sample.
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http://dx.doi.org/10.1016/j.foodchem.2020.128200DOI Listing
March 2021

On-line enrichment of N-glycans by immobilized metal-affinity monolith for capillary electrophoresis analysis.

Anal Chim Acta 2020 Oct 12;1134:1-9. Epub 2020 Aug 12.

Université Paris-Saclay, CNRS, Institut Galien Paris Saclay, 92296, Châtenay-Malabry, France; Institut Universitaire de France (IUF), France.

A novel N-glycan enrichment strategy is presented using unexpected but strong interactions between the sulfonate groups brought by the fluorescent dye of glycans and the Zr modified poly(ethylene glycol methacrylate phosphate (EGMP)-co-acrylamide (AM)-co-bis-acrylamide (BAA)) monolith. The poly (EGMP-co-AM-co-BAA) monolith was synthesized via ultraviolet (UV) irradiation and then functionalized with Zr. The obtained monolith was characterized with scanning electron microscopy and mercury intrusion porosimetry. Large through-pores and a continuous skeleton with high permeability were observed. The N-glycans were labeled with the 1-aminopyrene-3, 6, 8-trisulfonic acid (APTS) and enriched by the Zr modified monolith through IMAC interaction. This enrichment step was then coupled off-line to capillary electrophoresis (CE) separation with laser induced fluorescence (LIF) detection. Successful preconcentration of the APTS labeled maltooligosaccharide ladder was achieved under optimized conditions. Enrichment factors obtained for the maltooligosaccharides ranged from 9 to 24 with RSDs from 2.0% to 9.2% (n = 3). Moreover, very good repeatabilities (<6.7%) were obtained for glucose oligomers (4-15 glucose units) corresponding to sizes expected for N-glycans, demonstrating the great potential of this Zr modified monolith to enrich APTS labeled glycans from N-glycoproteins. The proposed method was then successfully applied for the enrichment of N-glycans released from Ribonuclease B, in which case all five expected oligomannose glycans (Man 5 to Man 9) were successfully enriched. Thanks to the advantage of the method to enrich selectively APTS-glycans compared to the commercial SPE columns composed of HILIC or PGC materials, the first proof of concept of on-line enrichment coupled to CE-LIF separation was demonstrated for maltooligosaccharides as well.
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http://dx.doi.org/10.1016/j.aca.2020.08.002DOI Listing
October 2020

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Anal Chem 2020 10 1;92(20):14103-14112. Epub 2020 Oct 1.

Division of Systems Biomedicine and Pharmacology, Leiden Academic Centre for Drug Research, Leiden University, 2311 G Leiden, The Netherlands.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (μ) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The μ was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the μ for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.
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http://dx.doi.org/10.1021/acs.analchem.0c03129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581015PMC
October 2020

Rapid Determination of Full and Empty Adeno-Associated Virus Capsid Ratio by Capillary Isoelectric Focusing.

Curr Mol Med 2020 ;20(10):814-820

Sciex, Brea, CA, United States.

Adeno-associated virus (AAV) is one of the most promising gene transfer vector types featuring long-term gene expression and low toxicity. The lack of pathogenicity and the availability of many serotypes augmented the applicability of AAV virions in gene therapy applications. The recombinant AAV capsid includes the therapeutic protein-coding transgene as well as a promoter to initiate translation and a poly A sequence portion for stabilization. Current AAV manufacturing technologies, however, cannot guarantee the generation of only full capsids, i.e., including the entire required genome. Partially filled and empty capsids are also part of the product, decreasing in this way the efficacy and safety upon clinical translation. Therefore, rapid, accurate and QC friendly analysis of the full and empty capsid ratio is of high importance during AAV vector manufacturing and release testing. In this paper, an automated capillary isoelectric focusing technique is introduced, readily applicable in the biopharmaceutical industry for fast and efficient determination of the full and empty capsid ratio. The method also reveals information about the proportion of partially filled capsids. For higher resolution (<0.1 pI unit), mixtures of wide and narrow range ampholytes were utilized. The isoelectric point and peak area percentage reproducibility (RSD) of the mixed ampholyte assay were as low as 1.67% and 2.45 %, respectively, requiring only 65 nL of sample volume per injection.
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http://dx.doi.org/10.2174/1566524020666200915105456DOI Listing
January 2020

Recent Advances in the Analysis Full/Empty Capsid Ratio and Genome Integrity of Adeno-associated Virus (AAV) Gene Delivery Vectors.

Curr Mol Med 2020 ;20(10):806-813

Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Egyetem u. 10, Hungary.

Adeno-associated virus (AAV) is one of the most promising viral gene delivery vectors with long-term gene expression and disease correction, featuring high efficiency and excellent safety in human clinical trials. During the production of AAV vectors, there are several quality control (QC) parameters that should be rigorously monitored to comply with clinical safety and efficacy. This review gives a short summary of the most frequently used AVV production and purification methods, focusing on the analytical techniques applied to determine the full/empty capsid ratio and the integrity of the encapsidated therapeutic DNA of the products.
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http://dx.doi.org/10.2174/1566524020999200730181042DOI Listing
January 2020

N-Glycosylation Alteration of Serum and Salivary Immunoglobulin a Is a Possible Biomarker in Oral Mucositis.

J Clin Med 2020 Jun 5;9(6). Epub 2020 Jun 5.

Institute of Pathology, Faculty of Medicine, University of Szeged, Állomás utca 1., H-6725 Szeged, Hungary.

Background: Oral and enteral mucositis due to high-dose cytostatic treatment administered during autologous and allogeneic stem-cell transplantation increases mortality. Salivary secretory immunoglobulin A (sIgA) is a basic pillar of local immunity in the first line of defense. Altered salivary sialoglycoprotein carbohydrates are important in the pathologies in the oral cavity including inflammation, infection and neoplasia. Therefore, we assessed whether changes in the salivary and serum IgA glycosylation correlated with development and severity of oral mucositis.

Methods: Using capillary electrophoresis, comparative analysis of serum and salivary IgA total N-glycans was conducted in 8 patients with autologous peripheral stem-cell transplantation (APSCT) at four different stages of transplantation (day -3/-7, 0, +7, +14) and in 10 healthy controls.

Results: Fourteen out of the 31 structures identified in serum and 6 out of 38 in saliva showed significant changes upon transplantation compared with the control group. Only serum core fucosylated, sialylated bisecting biantennary glycan (FA2BG2S2) showed significant differences between any two stages of transplantation (day -3/-7 and day +14; = 0.0279).

Conclusion: Our results suggest that changes in the serum IgA total N-glycan profile could serve as a disease-specific biomarker in patients undergoing APSCT, while analysis of salivary IgA N-glycan reflects the effect of APSCT on local immunity.
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http://dx.doi.org/10.3390/jcm9061747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355945PMC
June 2020

Diabetes-specific Modulation of Peripheral Blood Gene Expression Signatures in Colorectal Cancer.

Curr Mol Med 2020 ;20(10):773-780

Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Faculty of Medicine, Semmelweis University, Budapest, Hungary.

Background: Type 2 diabetes (T2DM) and colorectal cancer (CRC) are both known to modulate gene expression patterns in peripheral blood leukocytes (PBLs).

Objective: As T2DM has been shown to increase the incidence of CRC, we were prompted to check whether diabetes affects mRNA signatures in PBLs isolated from CRC patients.

Methods: Twenty-two patients were recruited to the study and classified into four cohorts (healthy controls; T2DM; CRC; CRC and T2DM). Relative expression levels of 573 cell signaling gene transcripts were determined by reverse transcription real-time PCR assays run on low-density OpenArray platforms. Enrichment analysis was performed with the g:GOSt profiling tool to order differentially expressed genes into functional pathways.

Results: 49 genes were found to be significantly up- or downregulated in tumorous diabetic individuals as compared to tumor-free diabetic controls, while 11 transcripts were differentially regulated in patients with CRC versus healthy, tumor-free and nondiabetic controls. Importantly, these gene sets were completely distinct, implying that diabetes exerts a profound influence on the transcription of signaling genes in CRC. The top 5 genes showing the most significant expression differences in both contexts were PCK2, MAPK9, CCND1, HMBS, TLR3 (p≤0.0040) and CREBBP, PPIA, NFKBIL1, MDM2 and SELPLG (p≤0.0121), respectively. Functional analysis revealed that most significantly affected pathways were cytokine, interleukin and PI3K/Akt/mTOR signaling cascades as well as mitotic regulation.

Conclusion: We propose that differentially expressed genes listed above might be potential biomarkers of CRC and should be studied further on larger patient groups. Diabetes might promote colorectal carcinogenesis by impairing signaling pathways in PBLs.
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http://dx.doi.org/10.2174/1566524020666200504084626DOI Listing
January 2020

Vaccine Plasmid Topology Monitoring by Capillary Gel Electrophoresis.

Curr Mol Med 2020 ;20(10):798-805

Pfizer, Chesterfield, Missouri, United States.

Background: Plasmid DNA has been widely used in vaccination as well as in cell and gene therapy. It exists in multiple isoforms, including supercoiled, nicked or open circular and linear forms. Regulatory agencies recommend having more than 80% of the supercoiled isoform for the bulk release of plasmid products; thus, it should be analyzed accordingly.

Methods And Results: The traditional analysis method for plasmid DNA is agarose gel electrophoresis. However, due to time-consuming manual sample loading, visualization, and data analysis, it has limitations in obtaining consistently quantitative results. In this short communication, we introduced a fast, sensitive, and robust plasmid analysis method using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). CGE-LIF analysis of the supercoiled isoform and its open circular counterpart was completed in 20 minutes with excellent sensitivity by using a common fluorescent DNA binding dye. The advantage of the method was demonstrated by the purity analysis of two large plasmids (7 kb and 10 kb). The fully automated sample loading, separation and data analysis featured enhanced assay repeatability and ease of quantitation over agarose gel electrophoresis.

Conclusion: As a worked example, analysis of plasmid samples treated at elevated temperature during an accelerated stability test also demonstrated the applicability of CGE-LIF to monitor plasmid topology and possible degradation.
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http://dx.doi.org/10.2174/1566524020666200427110452DOI Listing
January 2020

Modeling of the Desialylated Human Serum N-glycome for Molecular Diagnostic Applications in Inflammatory and Malignant Lung Diseases.

Curr Mol Med 2020 ;20(10):765-772

Horvath Csaba Memorial Laboratory of Bioseparation Sciences, Doctoral School of Molecular Medicine, Research Center for Molecular Medicine, Faculty of Medicine, University of Debrecen, Egyetem ter 1, Debrecen, Hungary.

Background: Immunoglobulin G and A, transferrin, haptoglobin and alpha-1- antitrypsin represent approximately 85% of the human serum glycoproteome and their N-glycosylation analysis may lead to the discovery of important molecular disease markers. However, due to the labile nature of the sialic acid residues, the desialylated subset of the serum N-glycoproteome has been traditionally utilized for diagnostic applications.

Objective: Creating a five-protein model to deconstruct the overall N-glycosylation fingerprints in inflammatory and malignant lung diseases.

Methods: The N-glycan pool of human serum and the five high abundant serum glycoproteins were analyzed. Simultaneous endoglycosidase/sialidase digestion was followed by fluorophore labeling and separation by CE-LIF to establish the model. Pooled serum samples from patients with COPD, lung cancer (LC) and their comorbidity were all analyzed.

Results: Nine significant (>1%) asialo-N-glycan structures were identified both in human serum and the standard protein mixture. The core-fucosylated-agalacto-biantennary glycan differentiated COPD and LC and both from the control and the comorbidity groups. Decrease in the core-fucosylated-agalacto-biantennary-bisecting, monogalacto and bigalacto structures differentiated all disease groups from the control. The significant increase of the fucosylated-galactosylated-triantennary structure was highly specific for LC, to a medium extent for COPD and a lesser extent for comorbidity. Also, some increase in the afucosylated-galactosylated-biantennary structure in all three disease types and afucosylated-galactosylated-triantennary structures in COPD and LC were observed in comparison to the control group.

Conclusion: Our results suggested that changes in the desialylated human serum Nglycome hold glycoprotein specific molecular diagnostic potential for malignant and inflammatory lung diseases, which can be modeled with the five-protein mixture.
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http://dx.doi.org/10.2174/1566524020666200422085316DOI Listing
January 2020

Multilevel Characterization of Antibody-Ligand Conjugates by CESI-MS.

Curr Mol Med 2020 ;20(10):789-797

Horvath Csaba Memorial Laboratory of Bioseparation Sciences, Research Centre for Molecular Medicine, Doctoral School of Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.

Aim: To demonstrate the capabilities of our new capillary electrophoresis - mass spectrometry method, which facilitates highly accurate relative quantitation of modification site occupancy of antibody-ligand (e.g., antibody-drug) conjugates.

Background: Antibody-drug conjugates play important roles in medical discovery for imaging and therapeutic intervention. The localization and stoichiometry of the conjugation can affect the orientation, selectivity, specificity, and strength of molecular interactions, influencing biochemical function.

Objective: To demonstrate the option to analyze the localization and stoichiometry of antibody-ligand conjugates by using essentially the same method at all levels including ligand infusion, peptide mapping, as well as reduced and intact protein analysis.

Materials And Methods: Capillary electrophoresis coupled with electrospray ionization mass spectrometry was used to analyze the antibody-ligand conjugates.

Results: We identified three prevalent ligand conjugation sites with estimated stoichiometries of 73, 14, and 6% and an average ligand-antibody ratio of 1.37, illustrating the capabilities of CE-ESI-MS for rapid and efficient characterization of antibody-drug conjugates.

Conclusion: The developed multilevel analytical method offers a comprehensive way to determine the localization and stoichiometry of antibody-drug conjugates for molecular medicinal applications. In addition, a significant advantage of the reported approach is the small, hydrophilic, unmodified peptides well separated from the neutrals, which is not common with other liquid phase separation methods such as LC.
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http://dx.doi.org/10.2174/1566524020666200415095830DOI Listing
January 2020

N-glycomic Analysis of Z(IgA1) Partitioned Serum and Salivary Immunoglobulin A by Capillary Electrophoresis.

Curr Mol Med 2020 ;20(10):781-788

Horvath Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Doctoral School for Medicine, Faculty of Medicine, University of Debrecen, Hungary.

Aims: Application of capillary electrophoresis with laser induced fluorescence detection (CE-LIF) to identify the N-glycosylation structures of serum and saliva IgA from healthy controls and patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions.

Background: Altered N-glycosylation of body fluid glycoproteins can be an effective indicator of most inflammatory processes. Immunoglobulin A (IgA) is the second highest abundant immunoglobulin and has a major role in the immune-defense against potential pathogen attacks. While IgA is abundant in serum, secretory immunoglobulin A (sIgA) is one of the most prevalent proteins in mucosal surfaces, such as in saliva.

Objective: Our aim was to investigate the changes of IgA glycosylation in serum and saliva as a response to an administered cytostatic treatment in patients with malignant hematological disorders.

Methods: Capillary electrophoresis with laser induced fluorescent detection (CE-LIF) was used to analyze the N-glycosylation profiles of Z(IgA1) partitioned immunoglobulin A in pooled serum and saliva of 10 control subjects and 8 patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions.

Results: Eight of 31 and four of 38 N-glycans in serum and saliva, respectively, showed significant (p<0.05) differences upon comparison to the control group. Thirteen glycans were present in the saliva but not in the serum, on the other hand, six structures were found in the serum samples not present in the saliva.

Conclusion: The developed Z(IgA1) partitioning and the high resolution CE-LIF based glyocoanalytical methods provided an efficient and sensitive workflow to detect and monitor IgA glycosylation alterations in serum and saliva with the scope for widespread molecular medicinal use.
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http://dx.doi.org/10.2174/1566524020666200413114151DOI Listing
January 2020

Glycoprotein biomarkers and analysis in chronic obstructive pulmonary disease and lung cancer with special focus on serum immunoglobulin G.

Clin Chim Acta 2020 Jul 31;506:204-213. Epub 2020 Mar 31.

University of Pannonia, 10 Egyetem Street, Veszprem 8200, Hungary; Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Centre for Molecular Medicine, Faculty of Medicine, University of Debrecen, 98 Nagyerdei Krt, Debrecen 4032, Hungary.

Chronic obstructive pulmonary disease (COPD) and lung cancer are two major diseases of the lung with high rate of mortality, mostly among tobacco smokers. The glycosylation patterns of various plasma proteins show significant changes in COPD and subsequent hypoxia, inflammation and lung cancer, providing promising opportunities for screening aberrant glycan structures contribute to early detection of both diseases. Glycoproteins associated with COPD and lung cancer consist of highly sialylated N-glycans, which play an important role in inflammation whereby hypoxia leads to accumulation of sialyl Lewis A and X glycans. Although COPD is an inflammatory disease, it is an independent risk factor for lung cancer. Marked decrease in galactosylation of plasma immunoglobulin G (IgG) together with increased presence of sialic acids and more complex highly branched N-glycan structures are characteristic for COPD and lung cancer. Numerous glycan biomarkers have been discovered, and analysis of glycovariants associated with COPD and lung cancer has been carried out. In this paper we review fundamental glycosylation changes in COPD and lung cancer glycoproteins, focusing on IgG to provide an opportunity to distinguish between the two diseases at the glycoprotein level with diagnostic value.
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http://dx.doi.org/10.1016/j.cca.2020.03.041DOI Listing
July 2020

N-glycosylation of blood coagulation factor XIII subunit B and its functional consequence.

J Thromb Haemost 2020 06 15;18(6):1302-1309. Epub 2020 Apr 15.

Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.

Background: The protective/inhibitory B subunits of coagulation factor XIII (FXIII-B) is a ~80 kDa glycoprotein containing two N-glycosylation sites. Neither the structure nor the functional role of the glycans on FXIII-B has been explored.

Objective: To reveal the glycan structures linked to FXIII-B, to design a method for deglycosylating the native protein, to find out if deglycosylation influences the dimeric structure of FXIII-B and its clearance from the circulation.

Methods: Asparagine-linked carbohydrates were released from human FXIIII-B by PNGase F digestion. The released N-linked oligosaccharides were fluorophore labeled and analyzed by capillary electrophoresis. Structural identification utilized glycan database search and exoglycosidase digestion based sequencing. The structure of deglycosylated FXIII-B was investigated by gel filtration. The clearance of deglycosylated and native FXIII-B from plasma was compared in FXIII-B knock out mice.

Results: PNGase F completely removed N-glycans from the denatured protein. Deglycosylation of the native protein was achieved by repeated digestion at elevated PNGase F concentration. The total N-glycan profile of FXIII-B featured nine individual structures; three were fucosylated and each structure contained at least one sialic acid. Deglycosylation did not change the native dimeric structure of FXIII-B, but accelerated its clearance from the circulation of FXIII-B knock out mice.

Conclusion: Characterization of the glycan moieties attached to FXIII-B is reported for the first time. Complete deglycosylation of the native protein was achieved by a deglycosylation workflow. The associated glycan structure is not required for FXIII-B dimer formation, but it very likely prolongs the half-life of FXIII-B in the plasma.
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http://dx.doi.org/10.1111/jth.14792DOI Listing
June 2020

The Effect of Temperature in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis of Protein Therapeutics.

Anal Chem 2020 03 18;92(5):4023-4028. Epub 2020 Feb 18.

Horváth Csaba Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, Doctoral School of Molecular Medicine, University of Debrecen, Debrecen, Hungary.

The temperature-dependent migration of molecular weight protein size standards and several biotherapeutic proteins were studied in sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) in the interval from 15 to 60 °C using borate cross-linked dextran sieving matrix. Arrhenius plots were generated to calculate the respective activation energy values for the various solute molecules. SDS-CGE analysis of the biotherapeutic protein test mixture revealed no correlation between the activation energy requirement of the different species and their molecular weights, emphasizing the importance of separation temperature optimization to obtain high resolution between the solute molecules of interest. In contrast, the molecular weight protein size ladder ranging from 10 to 225 kDa, built from the same polypeptide blocks with no post-translational and other modifications, showed predictable activation energy requirement. The electrophoretic mobility of the SDS-protein complexes was found to be the function of the reciprocal sixth root of the molecular weight (), implying cylindrical conformation.
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http://dx.doi.org/10.1021/acs.analchem.9b05566DOI Listing
March 2020

[The potential role of proteomic and glycomic biomarkers in chronic obstructive pulmonary disease diagnostics].

Orv Hetil 2020 Jan;161(4):123-128

Semmelweis Tagkórház, Tüdőgyógyászati Osztály, Borsod-Abaúj-Zemplén Megyei Központi Kórház és Egyetemi Oktatókórház Miskolc, Csabai kapu 9-11., 3529.

Chronic obstructive pulmonary disease (COPD) is worldwide a significant representative of morbidity and mortality statistics. COPD is a preventable and treatable disease and smoking is the main risk factor of disease development. Prevention is crucial, but it has its limitations, so risk estimation and early non-invasive diagnostics are essential to decrease COPD mortality. Although diagnostic techniques are evolving, the perfect screening tool is lacking. Discovery of properly sensitive and specific biomarkers is important. They could be effective diagnostic, differential diagnostic, phenotyping and prognostic tools to clinicians. The manuscript is focusing on recently discovered potential protein and glycan biomarkers for COPD. Orv Hetil. 2020; 161(4): 123-128.
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http://dx.doi.org/10.1556/650.2020.31640DOI Listing
January 2020

Comparative analysis of the human serum N-glycome in lung cancer, COPD and their comorbidity using capillary electrophoresis.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 Jan 12;1137:121913. Epub 2019 Dec 12.

Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Doctoral School of Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Hungary. Electronic address:

Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) are prevalent ailments with a great challenge to distinguish them based on symptoms only. Since they require different treatments, it is important to find non-invasive methods capable to readily diagnose them. Moreover, COPD increases the risk of lung cancer development, leading to their comorbidity. In this pilot study the N-glycosylation profile of pooled human serum samples (90 patients each) from lung cancer, COPD and comorbidity (LC with COPD) patients were investigated in comparison to healthy individuals (control) by capillary gel electrophoresis with high sensitivity laser-induced fluorescence detection. Sample preparation was optimized for human serum samples introducing a new temperature adjusted denaturation protocol to prevent precipitation and increased endoglycosidase digestion time to assure complete removal of the N-linked carbohydrates. The reproducibility of the optimized method was <3.5%. Sixty-one N-glycan structures were identified in the pooled control human serum sample and the profile was compared to pooled lung cancer, COPD and comorbidity of COPD with lung cancer patient samples. One important finding was that no other sugar structures were detected in any of the patient groups, only quantitative differences were observed. Based on this comparative exercise, a panel of 13 N-glycan structures were identified as potential glycobiomarkers to reveal significant changes (>33% in relative peak areas) between the pathological and control samples. In addition to N-glycan profile changes, alterations in the individual N-glycan subclasses, such as total fucosylation, degree of sialylation and branching may also hold important glycobiomarker values.
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http://dx.doi.org/10.1016/j.jchromb.2019.121913DOI Listing
January 2020

Recent advances in glycoinformatic platforms for glycomics and glycoproteomics.

Curr Opin Struct Biol 2020 06 23;62:56-69. Epub 2019 Dec 23.

Institute for Glycomics, Griffith University, Gold Coast, QLD, Australia. Electronic address:

Protein glycosylation is the most complex and prevalent post-translation modification in terms of the number of proteins modified and the diversity generated. To understand the functional roles of glycoproteins it is important to gain an insight into the repertoire of oligosaccharides present. The comparison and relative quantitation of glycoforms combined with site-specific identification and occupancy are necessary steps in this direction. Computational platforms have continued to mature assisting researchers with the interpretation of such glycomics and glycoproteomics data sets, but frequently support dedicated workflows and users rely on the manual interpretation of data to gain insights into the glycoproteome. The growth of site-specific knowledge has also led to the implementation of machine-learning algorithms to predict glycosylation which is now being integrated into glycoproteomics pipelines. This short review describes commercial and open-access databases and software with an emphasis on those that are actively maintained and designed to support current analytical workflows.
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http://dx.doi.org/10.1016/j.sbi.2019.11.009DOI Listing
June 2020

Preface.

Authors:
András Guttman

Curr Mol Med 2019 ;20(1)

Horváth Csaba Memorial Laboratory of Bioseparation Sciences, University of Debrecen Debrecen, Hungary.

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http://dx.doi.org/10.2174/156652402001191206100122DOI Listing
October 2020

Bioseparations - 2019.

Authors:
Andras Guttman

Electrophoresis 2019 12;40(23-24):3035

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http://dx.doi.org/10.1002/elps.201970204DOI Listing
December 2019

Expanding the capillary electrophoresis-based glucose unit database of the GUcal app.

Glycobiology 2020 05;30(6):362-364

Translational Glycomics Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, 10 Egyetem, Veszprem 8200, Hungary.

GUcal is a standalone application for automatically calculating the glucose unit (GU) values for separated N-glycan components of interest in an electropherogram and suggests their tentative structures by utilizing an internal database. We have expanded the original database of GUcal by integrating all publicly available capillary electrophoresis (CE) data in the GlycoStore collection (https://www.glycostore.org) and with in-house measured GU values. The GUcal app is freely available online (https://www.gucal.hu) and readily facilitates CE-based high throughput GU value determination for first line structural elucidation.
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http://dx.doi.org/10.1093/glycob/cwz102DOI Listing
May 2020

High Throughput Multiplex SNP-analysis in Chronic Obstructive Pulmonary Disease and Lung Cancer.

Curr Mol Med 2020 ;20(3):185-193

Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary.

Background: A number of human inflammatory diseases and tumors have been shown to cause alterations in the glycosylation pattern of plasma proteins in a specific manner. These highly variable and versatile post-translational modifications finetune protein functions by influencing sorting, folding, enzyme activity and subcellular localization. However, relatively little is known about regulatory factors of this procedure and about the accurate causative connection between glycosylation and disease.

Objective: The aim of the present study was to investigate whether certain single nucleotide polymorphisms (SNPs) in genes encoding glycosyltransferases and glycosidases could be associated with elevated risk for chronic obstructive pulmonary disease (COPD) and lung adenocarcinoma.

Methods: A total of 32 SNPs localized in genes related to N-glycosylation were selected for the association analysis. Polymorphisms with putative biological functions (missense or regulatory variants) were recruited. SNPs were genotyped by a TaqMan OpenArray platform. A single base extension-based method in combination with capillary gel electrophoresis was used for verification.

Results: The TaqMan OpenArray approach provided accurate and reliable genotype data (global call rate: 94.9%, accuracy: 99.6%). No significant discrepancy was detected between the obtained and expected genotype frequency values (Hardy-Weinberg equilibrium) in the healthy control sample group in case of any SNP confirming reliable sampling and genotyping. Allele frequencies of the rs3944508 polymorphism localized in the 3' UTR of the MGAT5 gene significantly differed between the sample groups compared.

Conclusion: Our results suggest that the rs34944508 SNP might modulate the risk for lung cancer by influencing the expression of MGAT5. This enzyme catalyzes the addition of N-acetylglucosamine (GlcNAc) in beta 1-6 linkage to the alpha-linked mannose of biantennary N-linked oligosaccharides, thus, increasing branching that is the characteristic of invasive malignancies.
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http://dx.doi.org/10.2174/1566524019666191017123446DOI Listing
January 2020

Sample Preparation Scale-Up for Deep N-glycomic Analysis of Human Serum by Capillary Electrophoresis and CE-ESI-MS.

Mol Cell Proteomics 2019 12 18;18(12):2524-2531. Epub 2019 Oct 18.

MTA-PE Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, 10 Egyetem Street, Veszprem, 8200, Hungary

We introduce an efficient sample preparation workflow to facilitate deep N-glycomics analysis of the human serum by capillary electrophoresis with laser induced fluorescence (CE-LIF) detection and to accommodate the higher sample concentration requirement of electrospray ionization mass spectrometry connected to capillary electrophoresis (CE-ESI-MS). A novel, temperature gradient denaturing protocol was applied on amine functionalized magnetic bead partitioned glycoproteins to circumvent the otherwise prevalent precipitation issue. During this process, the free sugar content of the serum was significantly decreased as well, accommodating enhanced PNGase F mediated release of the -linked carbohydrates. The liberated oligosaccharides were tagged with aminopyrene-trisulfonate, utilizing a modified evaporative labeling protocol. Processing the samples with this new workflow enabled deep CE-LIF analysis of the human serum N-glycome and provided the appropriate amount of material for CE-ESI-MS analysis in negative ionization mode.
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http://dx.doi.org/10.1074/mcp.TIR119.001669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885710PMC
December 2019