Characterization of N-Glycans in donkey milk lactoferrin by high resolution mass spectrometry

Within the frame of our research line directed to the investigation of donkey milk proteins [1] we have previously reported the sequence characterization and glycosylation sites identification of donkey milk lactoferrin [2]. Lactoferrin, member of the transferrin family, is one of the most important glycoproteins [3] present in the whey protein fraction of milk. Its function is modulated by both the polypeptide chain and its glycosylation [4]. We report now a comprehensive glycosylation profile of donkey lactoferrin, isolated by ion exchange chromatography from an individual milk sample, by means of chymotryptic digestion, TiO2 and Hydrophilic Interaction Liquid Chromatography (HILIC) enrichment, reversed-phase high performance liquid chromatography, electrospray mass spectrometry and high collision dissociation fragmentation. To identify the glycopeptides, mass spectral data were processed by an in-house developed software (MassAI Bioinformatics) [5]. This study revealed that in donkey milk lactoferrin most of the N-glycans identified are neutral complex/hybrid. Actually, 10 neutral non-fucosylated hybrid/complex N-glycans and 4 neutral fucosylated hybrid/complex N-glycans were found. In addition, 2 high mannose N-glycans, 4 sialylated fucosylated hybrid/complex N-glycans and 6 sialylated non-fucosylated N-glycans, one of which containing N-glycolylneuramin acid (Neu5Gc), were found. References: [1] Cunsolo V, Muccilli V, Saletti R, Foti S (2011) Applications of mass spectrometry techniques in the investigation of milk proteome. Eur J Mass Spectrom 17(4):305-320 [2] Gallina S, Cunsolo V, Saletti R, Muccilli V, Di Francesco A, Foti S, Lorentzen A M, Roepstorff P (2016) Sequence characterization and glycosylation sites identification of donkey milk lactoferrin by multiple enzyme digestions and mass spectrometry. Amino Acids. doi: 10.1007/s00726-016-2209-0 [3]Hutchens TW, Lönnerdal B, Rumball S (1994) Lactoferrin structure and function - remaining questions, methodological considerations and future directions. In:Hutchens TW, Rumball SV, Lönnerdal B (eds) Lactoferrin: Structure and Function. Springer, New York, pp 287-291 [4] Barboza M, Pinzon J, Wickramasinghe S, Froehlich JW, Moeller I, Smilowitz JT, Ruhaak LR, Huang J, Löennerdal B, German JB, Medrano JF, Weimer BC, Lebrilla CB (2012) Glycosylation of human milk lactoferrin exhibits dynamic changes during early lactation enhancing its role in pathogenic bacteria-host interactions. Mol Cell Proteomics 11(6):M111.015248 [5]MassAI-Bioinformatics. MassAI. At <http://massai.dk/index.html>