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Updated: Jan 19, 2026
Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications
Published on: May 18, 2017
Effect of the elapsed time between sampling and formalin fixation on the N-glycosylation profile of mouse tissue
Boglarka Donczo1, Gabor Kiraly2, Andras Guttman1,3
1Horváth Csaba Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Abstract:
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.
Insights
Investigating the impact of sample handling time on N-glycosylation profiles in mouse tissues reveals that up to two hours of room temperature storage does not alter complex carbohydrates but decreases linear glucose oligomers and high mannose glycans.
Area of Science:
- Biochemistry
- Molecular Biology
- Glycomics
Background:
- Formalin-fixed, paraffin-embedded (FFPE) samples are crucial for histological studies and contain valuable molecular diagnostic information.
- While the FFPE process itself doesn't affect N-glycan moieties, the impact of pre-fixation storage time on N-glycosylation profiles remains uncharacterized.
Purpose of the Study:
- To investigate the effect of elapsed time between sampling and fixation on N-glycosylation profiles in mouse tissues.
- To assess changes in N-glycan profiles of lung tissue stored at room temperature for up to two hours post-sampling.
Main Methods:
- N-glycan profiling was performed on various mouse tissues (lung, brain, heart, spleen, liver, kidney, intestine).
- Lung tissue samples were analyzed at zero, one, and two hours post-sampling.
- Glycoprotein extraction using RIPA buffer, PNGase F digestion, fluorophore labeling, and capillary electrophoresis-laser induced fluorescent detection (CE-LIF) were employed.
- Deep glycomic analysis involved carbohydrate profiling and exoglycosidase treatment.
Main Results:
- Up to two hours of room temperature storage did not significantly alter the N-glycosylation profiles of complex carbohydrates in mouse lung tissue.
- A notable decrease in the abundance of linear glucose oligomers and high mannose type glycans was observed with increasing storage time.
- These findings suggest that while complex N-glycans are relatively stable, certain glycan structures are sensitive to pre-fixation delays.
Conclusions:
- Short delays (up to 2 hours) in fixation of mouse tissues do not broadly impact N-glycosylation profiles, particularly complex carbohydrates.
- The observed decrease in linear glucose oligomers and high mannose glycans indicates potential structural changes or degradation under prolonged room temperature storage.
- These results are critical for standardizing sample handling protocols in glycomic studies utilizing FFPE tissues for molecular diagnostics.
