Improved sample preparation for CE‐LIF analysis of plant N‐glycans

Abstract

In view of glycomics studies in plants, it is important to have sensitive tools that allow one to analyze and characterize the N-glycans present on plant proteins in different species. Earlier methods combined plant-based sample preparations with CE-LIF N-glycan analysis but suffered from background contaminations, often resulting in non-reproducible results. This publication describes a reproducible and sensitive protocol for the preparation and analysis of plant N-glycans, based on a combination of the 'in-gel release method' and N-glycan analysis on a multicapillary DNA sequencer. Our protocol makes it possible to analyze plant N-glycans starting from low amounts of plant material with highly reproducible results. The developed protocol was validated for different plant species and plant cells.

Comparison of VHH‐Fc antibody production in Arabidopsis thaliana,Nicotiana benthamiana and Pichia pastoris

“…N‐glycans of plant‐produced VHH‐Fcs were also analysed after deglycosylation with PNGase A according to Nagels et al . (). The N‐glycan composition was analysed by exoglycosidase digestions and determined on an ABI 3130 DNA sequencer (Applied Biosciences, Waltham, Massachusetts).…”

Combined N-Glycome and N-Glycoproteome Analysis of the Lotus japonicus Seed Globulin Fraction Shows Conservation of Protein Structure and Glycosylation in Legumes

“…The three most abundant N-glycan compositions on the Lotus seed globulins were GlcNAc 2 Man 3 Xyl 1 Fuc 1 GlcNAc 2 , Man 3 Xyl 1 Fuc 1 GlcNAc 2 , and Man 3 Xyl 1 GlcNAc 2 , which are similar to the abundant N-glycans in leaf tissues of other plant species . All pauci-mannosidic and complex structures from the Lotus seed globulins contained the plant-specific β1,2-xylose and 80% of these were α-1,3-core-fucosylated whereas the high mannose N - glycans were not fucosylated.…”

“…44 The three most abundant N-glycan compositions on the Lotus seed globulins were GlcNAc 2 Man 3 Xyl 1 Fuc 1 GlcNAc 2 , Man 3 Xyl 1 Fuc 1 GlcNAc 2 , and Man 3 Xyl 1 GlcNAc 2 , which are similar to the abundant N-glycans in leaf tissues of other plant species. 45 All pauci-mannosidic and complex structures from the Lotus seed globulins contained the plant-specific β1,2-xylose and 80% of these were α-1,3-core-fucosylated whereas the high mannose N-glycans were not fucosylated. In the context of food allergy, the high content of the plant-specific α-1,3-fucose and β-1,2-xylose determinants in the Lotus globulin fraction may be relevant for triggering allergic reactions either by recognition of the plant specific N-glycans by the immune system 21−25 and/or by altering the proteolytical degradation of proteins in the gastrointestinal tract.…”

Applying Glycomic Technology to Investigate the Consequences of Altered Glycosylation in Human ST3GAL5 Deficiency

“…These labeling/HPLC-based approaches provide a robust means of quantifying absolute glycan amount based on fluorescence yield and allow glycan structure to be inferred based on chromatographic retention and sequencing by glycosidase digestion. Improvements in the resolution, speed, and mass accuracy of mass spectrometers have facilitated direct structural analysis of glycans found in complex biological samples, making mass spectrometry (MS) an essential tool for glycomic analysis (22)(23)(24). However, compared to the other analytical approaches (HPLC, CE, and NMR), the quantitative nature of MS-based analysis has generally been limited to relative quantification, in which the contribution of the detected signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total glycan profile.…”