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001-es BibID:	BIBFORM122537
035-os BibID:	(Scopus)85197084238 (WoS)001259901900001
Első szerző:	Auer, Felicia
Cím:	Noncovalent Fluorophore Labeling of Proteins by Propidium Iodide in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis / Auer Felicia, Guttman Andras
Dátum:	2024
ISSN:	0003-2700
Megjegyzések:	Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis. As a key ingredient of the gel-buffer system, the oppositely migrating positively charged propidium ligand in migratio complexes with the SDS-proteins, therefore, supports in situ labeling during the electrophoretic separation process, not requiring any extra pre- or postcolumn derivatization step. A theoretical treatment is given to shed light on the basic principles of this novel online labeling process, also addressing the influence of propidium iodide on the electroosmotic flow, resulting in reduced retardation. The concept of propidium labeling in SDS-CGE was first demonstrated using a commercially available protein sizing ladder ranging from 6.5 to 200 kDa with different isoelectric points and post-translational modifications. Considering the increasing number of protein therapeutics on the market next, we focused on the labeling optimization of a therapeutic monoclonal antibody and its subunits, including the addition of the nonglycosylated heavy chain. Peak efficiency and resolution were compared between noncovalent and covalent labeling. The effect of ligand concentration on the effective and apparent electrophoretic mobility, the resulting peak area, and the resolution were all evaluated in view of the theoretical considerations. The best detection sensitivity for the intact monoclonal antibody was obtained by using 200 ?g/mL propidium iodide in the separation medium (LOD 2 ?g/mL, 1.35 ? 10-8 M) with excellent detection linearity over 3 orders of magnitude. On the other hand, the resolution between the biopharmaceutical protein test mixture components containing the intact and subunit fragments of the therapeutic monoclonal antibody was very good in the ligand concentration range of 50-200 ?g/mL, but using the local maximum at 100 ?g/mL for the nonglycosylated/glycosylated heavy chain pair is recommended. The figures of merit, including precision, sensitivity, detection linear range, and resolution for a sample mixture in hand, can be optimized by varying the propidium iodide concentration in the gel-buffer system, as demonstrated in this paper.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk
Megjelenés:	Analytical Chemistry. - [Epub ahead of print] (2024). -
További szerzők:	Guttman András (1954-) (vegyészmérnök)
Pályázati támogatás:	TUDFIN Egyéb
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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001-es BibID:	BIBFORM113960
035-os BibID:	(WoS)001043559400001 (Scopus)85166981215
Első szerző:	Auer, Felicia
Cím:	Size separation of sodium dodecyl sulfate-proteins by capillary electrophoresis in dilute and ultra-dilute dextran solutions / Felicia Auer, Andras Guttman
Dátum:	2023
ISSN:	0173-0835 1522-2683
Megjegyzések:	SDS capillary gel electrophoresis is a widely used in the biopharma and the biomedical fields for rapid size separation of proteins. However, very limited information is available on the use of dilute and ultra-dilute sieving matrices for SDS?protein analysis. Here, background electrolytes (BGEs) containing 1%? 0% dextran were used in borate-based BGE to separate a protein sizing ladder (PSL) ?225 kDa and the intact and subunit forms of a therapeutic monoclonal antibody (mAb). The separation performance for the PSL and mAb components differed significantly with decreasing dextran concentration. Ferguson and reptation plots were used to elucidate the separation mechanism. Highly diluted dextran solutions resulted in linear Ferguson plots for both solute types (cf. Ogston theory) in spite of this model assumes a rigid pore structure, thus cannot describe the separation mechanism in ultra-dilute polymer solutions with no reticulations. The saddle differences between the resolution of the PSL and the intact/subunit mAb forms in ultra-dilute dextran-borate matrices suggested the importance of shape selectivity, manifested by the adequate separation of the SDS covered intact as well as light and heavy chain subunits of the therapeutic mAb even at zero dextran concentration.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk borate capillary gel electrophoresis dextran SDS-proteins ultra-dilute polymer
Megjelenés:	Electrophoresis. - 44 : 19-20 (2023), p. 1607-1614. -
További szerzők:	Guttman András (1954-) (vegyészmérnök)
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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001-es BibID:	BIBFORM099680
035-os BibID:	(cikkazonosító)122497
Első szerző:	Auer, Felicia
Cím:	Recent advances in the analysis of human milk oligosaccharides by liquid phase separation methods / Auer Felicia, Jarvas Gabor, Guttman Andras
Dátum:	2021
ISSN:	1570-0232
Megjegyzések:	Human milk is a complex, dynamically changing biological fluid, which contains a large amount of nonconjugated 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.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk Human milk Oligosaccharides Analysis Chromatography Electrophoresis Mass spectrometry
Megjelenés:	Journal Of Chromatography B-Analytical Technologies In The Biomedical And Life Sciences. - 1162 (2021), p. 1-9. -
További szerzők:	Járvás Gábor (1982-) (vegyészmérnök) Guttman András (1954-) (vegyészmérnök)
Pályázati támogatás:	2018-2.1.17-T?ET-KR-2018-00010 Egyéb BIONANO_GINOP-2.3.2- 15-2016-00017 GINOP NN127062 OTKA TKP2020-IKA-07 OTKA UNKP-20-5 Egyéb
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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001-es BibID:	BIBFORM106825
035-os BibID:	(cikkazonosító)8192 (scopus)85143597041 (wos)000896340300001
Első szerző:	Torok Rebeka
Cím:	The Effect of Sample Glucose Content on PNGase F-Mediated N-Glycan Release Analyzed by Capillary Electrophoresis / Torok Rebeka, Auer Felicia, Farsang Robert, Jona Eszter, Jarvas Gabor, Guttman Andras
Dátum:	2022
ISSN:	1420-3049
Megjegyzések:	Protein therapeutics have recently gained high importance in general health care along with applied clinical research. Therefore, it is important to understand the structure?function relationship of these new generation drugs. Asparagine-bound carbohydrates represent an important critical quality attribute of therapeutic glycoproteins, reportedly impacting the efficacy, immunogenicity, clearance rate, stability, solubility, pharmacokinetics and mode of action of the product. In most instances, these linked N-glycans are analyzed in their unconjugated form after endoglycosidase-mediated release, e.g., PNGase F-mediated liberation. In this paper, first, N-glycan release kinetics were evaluated using our previously reported in-house produced 6His-PNGase F enzyme. The resulting deglycosylation products were quantified by sodium dodecyl sulfate capillary gel electrophoresis to determine the optimal digestion time. Next, the effect of sample glucose content was investigated as a potential endoglycosidase activity modifier. A comparative Michaelis-Menten kinetics study was performed between the 6His-PNGase F and a frequently employed commercial PNGase F product with and without the presence of glucose in the digestion reaction mixture. It was found that 1 mg/mL glucose in the sample activated the 6His-PNGase F enzyme, while did not affect the release efficiency of the commercial PNGase F. Capillary isoelectric focusing revealed subtle charge heterogeneity differences between the two endoglycosidases, manifested by the lack of extra acidic charge variants in the cIEF trace of the 6His-PNGase F enzyme, which might have possibly influenced the glucose-mediated enzyme activity differences.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk
Megjelenés:	Molecules. - 27 : 23 (2022), p. 1-8. -
További szerzők:	Auer, Felicia Farsang Róbert Jóna Eszter Járvás Gábor (1982-) (vegyészmérnök) Guttman András (1954-) (vegyészmérnök)
Pályázati támogatás:	UNKP-22-3-I Egyéb
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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