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 and are compatible with microwell-plate sampling, without the need for tedious centrifugation, precipitation, or sample-transfer steps. The versatility of this methodology was evaluated by analyzing glycoproteins derived from various plant sources using ultra-performance liquid chromatography (UPLC) analysis and further demonstrated through the activity analysis of four PNGase H(+) mutant variants.