Characterization And Comparison Of Ovotransferrin N-Glycans From Two Separate Sources Via Mass Spectrometry

Ovotransferrin is a group of N-glycosylated protein, accounting for12~13%of total avain egg white proteins. It possesses a wide range of biofunctions including antibacteria, antifungus, antivirus, anticancer and antioxidation. However, for now, very little is known about either the N-glycan structures of specific ovotransferrin or the N-glycan differences among ovotransferrin of various avian species.In this study, we prepared ovotransferrin from both chicken (Gallus domesticus) and pheasant (Phasianus colchicus) egg white, using the method of two-time ethanol precipitation. The N-glycans of purified chicken and peasant ovotransferrin were released by PNGase F, and followed by composition analysis via ESI-MS. For quantitative comparison of the N-glycans of chicken and pheasant ovotransferrin, the strategy of aniline stable isotopic labeling combined with ESI/MS was employed. Online HILIC-MS and MS/MS were also utilized to separate and reveal the structures of isomeric glycans from chicken and pheasant ovotransferrin as well as to compare the difference in abundance between the N-glycans found in both chicken and pheasant ovotransferrin. The results are described as follows,1. SDS-PAGE and MALDI-TOF-MS analysis show the molecular weights of chicken and peasant ovotransferrins are77284Da and76774Da, respectively. The purity of the two ovotransferrin is high enough to meet glycan structure analysis.2. In total,16N-glycans corresponding to12N-glycan compositions were found in chicken ovotrasnferrin, of which1is core structure (3.18%),12are complex type (91.40%) and3are hybrid type (5.42%); while21N-glycans corresponding to15N-glycan compositions were found in pheasant ovotransferrin, of which1is truncated structure (1.88%),1is core structure (6.26%),3are high mannose type (5.20%),10 are complex type (67.52%) and6are hybrid type (19.14%). All of the N-glycans founded in chicken and pheasant ovotransferrin are neutral and no fucosylated or sialylated glycans were observed.3. Ten N-glycans exist in both chicken and pheasant ovotransferrin and their abundance ratios were also demonstrated. This study provides a base for further investigation of species-specific ovotransferrin in glycobiology and the functional diversity between chicken and pheasant ovotransferrin.