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Rapid Sample Preparation Methodology for Plant N-Glycan Analysis Using Acid-Stable PNGase H+

The quantification of potentially allergenic carbohydrate motifs of plant and insect glycoproteins is increasingly important in biotechnological and agricultural applications as a result of the use of insect cell-based expression systems and transgenic plants. The need to analyze N-glycan moieties in a highly parallel manner inspired us to develop a quick N-glycan analysis method based on a recently discovered bacterial protein N-glycanase (PNGase H+). In contrast to the traditionally used PNGase A, which is isolated from almond seeds and only releases N-glycans from proteolytically derived glycopeptides, the herein implemented PNGase H+ allows for the release of N-glycans directly from the glycoprotein samples. Because PNGase H+ is highly active under acidic conditions, the consecutive fluorescence labeling step using 2-aminobenzamide (2AB) can be directly performed in the same mixture used for the enzymatic deglycosylation step. All sample handling and incubation steps can be performed in less than 4 h ...

Authors:   Ya M. Du; Tian Xia; Xiao Q. Gu; Ting Wang; Hong Y. Ma; Josef Voglmeir; Li Liu
Journal:   Journal of Agricultural and Food Chemistry
Year:   2015
DOI:   10.1021/acs.jafc.5b03633
Publication date:   23-Nov-2015
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