title: Rapid Sample Preparation Methodology for Plant N-Glycan Analysis Using Acid-Stable PNGase H+.
abstract: 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.
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, and ‡Department of Plant Pathology, Nanjing Agricultural University , Nanjing, Jiangsu 210095, People's Republic of China.
authors.forename: Ya M
authors.initials: YM
authors.lastname: Du
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, and ‡Department of Plant Pathology, Nanjing Agricultural University , Nanjing, Jiangsu 210095, People's Republic of China.
authors.forename: Tian
authors.initials: T
authors.lastname: Xia
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, and ‡Department of Plant Pathology, Nanjing Agricultural University , Nanjing, Jiangsu 210095, People's Republic of China.
authors.forename: Xiao Q
authors.initials: XQ
authors.lastname: Gu
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, and ‡Department of Plant Pathology, Nanjing Agricultural University , Nanjing, Jiangsu 210095, People's Republic of China.
authors.forename: Ting
authors.initials: T
authors.lastname: Wang
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, and ‡Department of Plant Pathology, Nanjing Agricultural University , Nanjing, Jiangsu 210095, People's Republic of China.
authors.forename: Hong Y
authors.initials: HY
authors.lastname: Ma
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, and ‡Department of Plant Pathology, Nanjing Agricultural University , Nanjing, Jiangsu 210095, People's Republic of China.
authors.forename: Josef
authors.initials: J
authors.lastname: Voglmeir
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, and ‡Department of Plant Pathology, Nanjing Agricultural University , Nanjing, Jiangsu 210095, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D055162:Biocatalysis; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis*
chemical_list: D001426:Bacterial Proteins; D006023:Glycoproteins; D011134:Polysaccharides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: N-linked glycosylation; PNGase; PNGase H+; Terriglobus; core fucosylation; plant glycan analysis