Molecular glycopathology by capillary electrophoresis: Analysis of the N‐glycome of formalin‐fixed paraffin‐embedded mouse tissue samples

Döncző, Boglárka; Szarka, Máté; Tóvári, József; Ostoros, Gyorgyi; Csánky, Eszter; Guttman, András

doi:10.1002/elps.201600558

Cited by 8 publications

(7 citation statements)

References 23 publications

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“…CE is a high-resolution method that can separate complex carbohydrate molecules while offering excellent sensitivity. This method, in conjunction with laser-induced fluorescence (LIF) detection, can reach detection limits in the femtomolar range [ 156 ] and is optimal for profiling fluorophore-labelled oligosaccharides [ 157 ]. Briefly, this analytical system used for carbohydrate profiling separates and analyses oligosaccharides released from glycoproteins.…”

Section: Glycomics Techniquesmentioning

confidence: 99%

Glycosylation Biomarkers Associated with Age-Related Diseases and Current Methods for Glycan Analysis

Paton

Suárez

Herrero

et al. 2021

IJMS

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Ageing is a complex process which implies the accumulation of molecular, cellular and organ damage, leading to an increased vulnerability to disease. In Western societies, the increase in the elderly population, which is accompanied by ageing-associated pathologies such as cardiovascular and mental diseases, is becoming an increasing economic and social burden for governments. In order to prevent, treat and determine which subjects are more likely to develop these age-related diseases, predictive biomarkers are required. In this sense, some studies suggest that glycans have a potential role as disease biomarkers, as they modify the functions of proteins and take part in intra- and intercellular biological processes. As the glycome reflects the real-time status of these interactions, its characterisation can provide potential diagnostic and prognostic biomarkers for multifactorial diseases. This review gathers the alterations in protein glycosylation profiles that are associated with ageing and age-related diseases, such as cancer, type 2 diabetes mellitus, metabolic syndrome and several chronic inflammatory diseases. Furthermore, the review includes the available techniques for the determination and characterisation of glycans, such as liquid chromatography, electrophoresis, nuclear magnetic resonance and mass spectrometry.

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Section: Glycomics Techniquesmentioning

confidence: 99%

Glycosylation Biomarkers Associated with Age-Related Diseases and Current Methods for Glycan Analysis

Paton

Suárez

Herrero

et al. 2021

IJMS

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“…4.2b,c) (Hennig et al 2016;Schwedler et al 2014a, b;Chen et al 2017;Huang et al 2017;Guttman et al 2015)], along with position and linkage of fucose (Fuc) [e.g., α1-6 core Fuc from α1-3/1-4 Fuc on antenna N-acetylglucosamine (GlcNAc) or α1-2 Fuc on galactose (Hennig et al 2016;Konze et al 2017;Thiesler et al 2016;Weiz et al 2016;Schwedler et al 2014a)], type and linkage of sialic acids (Sia) [e.g., N-acetylneuraminic acid (Neu5Ac) from N-glycolylneuraminic acid (Neu5Gc), see Fig. 4.3d (Abeln et al 2017), and α2-3 from α2-6 (Hennig et al 2016;Konze et al 2017;Thiesler et al 2016;Donczo et al 2017;Meininger et al 2016)], and linkage of galactose (Gal) [e.g., β1-3 Gal from β1-4 Gal (Konze et al 2017;Thiesler et al 2016;Schwedler et al 2014a;Muñoz et al 2019)]. This feature becomes especially advantageous when thinking about the importance of determining immunogenic α-Gal and Neu5Gc on glycoprotein therapeutics (Chung et al 2008;Teranishi et al 2002;Van der Linden et al 2000).…”

Section: Background: Principles Of Capillary (Gel) Electrophoresis (C(g)e)mentioning

confidence: 99%

Capillary (Gel) Electrophoresis-Based Methods for Immunoglobulin (G) Glycosylation Analysis

Cajic

Hennig

Burock³

et al. 2021

Experientia Supplementum

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The in-depth characterization of protein glycosylation has become indispensable in many research fields and in the biopharmaceutical industry. Especially knowledge about modulations in immunoglobulin G (IgG) N-glycosylation and their effect on immunity enabled a better understanding of human diseases and the development of new, more effective drugs for their treatment. This chapter provides a deeper insight into capillary (gel) electrophoresis-based (C(G)E) glycan analysis, addressing its impressive performance and possibilities, its great potential regarding real high-throughput for large cohort studies, as well as its challenges and limitations. We focus on the latest developments with respect to miniaturization and mass spectrometry coupling, as well as data analysis and interpretation. The use of exoglycosidase sequencing in combination with current C(G)E technology is discussed, highlighting possible difficulties and pitfalls. The application section describes the detailed characterization of N-glycosylation, utilizing multiplexed CGE with laser-induced fluorescence detection (xCGE-LIF). Besides a comprehensive overview on antibody glycosylation by comparing species-specific IgGs and human immunoglobulins A, D, E, G, and M, the chapter comprises a comparison of therapeutic monoclonal antibodies from different production cell lines, as well as a detailed characterization of Fab and Fc glycosylation. These examples illustrate the full potential of C(G)E, resolving the smallest differences in sugar composition and structure.

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“…For mouse lung tissue, the effect of the elapsed time between the actual sampling and the formalin fixation, paraffin embedding process has been investigated to understand its influence on N ‐glycosylation degradation in zero time point as well as after 1 and 2 h of room temperature storage. All other mouse tissue sample specimens were profiled for their N ‐glycosylation since different tissue sample types have unique N ‐glycan profiles, specific to that particular organ containing diverse proportions of high and low sialylated, high mannose as well as neutral carbohydrate structures in different size ranges, and interestingly a few low degree of polymerization glucose oligomers.…”

Section: Resultsmentioning

confidence: 99%

“…Our earlier results revealed that the N‐linked carbohydrates of glycoproteins with neutral, sialylated and high mannose structures were not affected by the formalin fixation and paraffin embedding process . Thus, it was considered that the fixation process archived the N‐glycan structures and compositions of the sample at the very moment it was prepared.…”

Section: Introductionmentioning

confidence: 98%

Effect of the elapsed time between sampling and formalin fixation on the N‐glycosylation profile of mouse tissue specimens

2019

Self Cite

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Formalin‐fixed, paraffin‐embedded (FFPE) samples are generally used for histology‐study, however, they also possess important molecular diagnostics information. While it has been reported that the N‐glycan moieties of glycoproteins is not affected by the FFPE process, no information is available about the effect of the elapsed time between sampling and fixation on the resulting N‐glycosylation profile. In this study, lung, brain, heart, spleen, liver, kidney, and intestine mouse tissue specimens were used for N‐glycan profiling analysis and the elapsed sampling time effect was investigated with the lung tissue. N‐glycan extraction from the tissue samples was performed by glycoprotein retrieval from the FFPE specimens using radioimmunoprecipitation assay (RIPA) buffer followed PNGase F digestion. The released oligosaccharides were fluorophore labeled and analyzed by capillary electrophoresis‐laser induced fluorescent detection (CE‐LIF). N‐glycosylation profiles of freshly collected lung‐tissue samples (zero time point), as well as 1 and 2 h after sampling were compared by carbohydrate profiling and exoglycosidase treatment based deep glycomic analysis. It was found that up to two hours of room temperature storage of tissue specimens apparently did not cause changes in the N‐glycosylation profiles of complex carbohydrates, but resulted in considerable decrease in the amount of linear glucose oligomers and high mannose type glycans present in the samples.

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Molecular glycopathology by capillary electrophoresis: Analysis of the N‐glycome of formalin‐fixed paraffin‐embedded mouse tissue samples

Cited by 8 publications

References 23 publications

Glycosylation Biomarkers Associated with Age-Related Diseases and Current Methods for Glycan Analysis

Glycosylation Biomarkers Associated with Age-Related Diseases and Current Methods for Glycan Analysis

Capillary (Gel) Electrophoresis-Based Methods for Immunoglobulin (G) Glycosylation Analysis

Effect of the elapsed time between sampling and formalin fixation on the N‐glycosylation profile of mouse tissue specimens

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