Comparison of chicken and pheasant ovotransferrin N-glycoforms via electrospray ionization mass spectrometry and liquid chromatography coupled with mass spectrometry.

Species-specific ovotransferrin features a highly conservative protein sequence, but it varies in the structure of the attached oligosaccharides, which may contribute to the differences observed in its bioactivity and nutritional value. Herein, chicken ovotransferrin (COT) and pheasant ovotransferrin (POT) isolated by repeated ethanol precipitation of egg white were digested with peptide N-glycosidase F to release N-glycans. The obtained N-glyans were isotopically labeled with aniline and analyzed via electrospray ionization mass spectrometry and online hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS). Relative quantitation based on isotopic aniline labeling and HILIC-MS/MS analysis revealed in detail the conspicuous difference between COT and POT in the abundance of their N-glycan compositions and isomers. In total, 16 COT N-glycans were observed, including 1 core structure (3.18%), 3 hybrid type (5.42%), and 12 complex type (91.40%), whereas 21 POT N-glycans were found, including 1 truncated structure (1.88%), 1 core structure (6.26%), 3 high mannose type (5.20%), 6 hybrid type (19.14%), and 10 complex type (67.52%). To our knowledge, this study is the first qualitative and quantitative comparison of COT and POT N-glycosylation patterns. These results suggest that POT has a different glycosylation pattern compared to that of COT and thus the effect of its glycosylation pattern on its bioactivity is worthy of further exploration.