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Site-specific glycosylation of donkey milk lactoferrin investigated by high-resolution mass spectrometry

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Abstract

A comprehensive monosaccharide composition of the N-glycans of donkey milk lactoferrin, isolated by ion exchange chromatography from an individual milk sample, was obtained by means of chymotryptic digestion, TiO2 and HILIC enrichment, reversed-phase high-performance liquid chromatography, electrospray mass spectrometry, and high collision dissociation fragmentation. The results obtained allowed identifying 26 different glycan structures, including high mannose, complex and hybrid N-glycans, linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476. Altogether, the N-glycan structures determined revealed that most of the N-glycans identified in donkey milk lactoferrin are neutral complex/hybrid. Indeed, 10 neutral non-fucosylated complex/hybrid N-glycans and 4 neutral fucosylated complex/hybrid N-glycans were found. In addition, two high mannose N-glycans, four sialylated fucosylated complex N-glycans and six sialylated non-fucosylated complex N-glycans, one of which containing N-glycolylneuraminic acid (Neu5Gc), were found. A comparison of the monosaccharide composition of the N-glycans of donkey milk lactoferrin with respect to that of human, bovine and goat milk lactoferrin is reported. Data are available via ProteomeXchange with identifier PXD004289.

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Acknowledgments

This work was supported by Grants from PO FERS 2007/13 4.1.2.A, project “Piattaforma regionale di ricerca translazionale per la salute”, CUP B65E12000570008 and from POR 2007/2013 project “BRIT” CUP E61D11000280007. The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD004289. We are grateful to Dr. D. Franchina and Dr. K. Torrisi (ASILAT srl farm, Milo, Catania) for the gift of the raw donkey milk.

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Authors and Affiliations

  1. Organic Mass Spectrometry Laboratory, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125, Catania, Italy

    Serafina Gallina, Rosaria Saletti, Vincenzo Cunsolo, Vera Muccilli & Salvatore Foti

  2. Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark

    Peter Roepstorff & Morten I. Rasmussen

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  1. Serafina Gallina
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  2. Rosaria Saletti
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  3. Vincenzo Cunsolo
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  5. Salvatore Foti
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  7. Morten I. Rasmussen
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Correspondence to Salvatore Foti.

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Gallina, S., Saletti, R., Cunsolo, V. et al. Site-specific glycosylation of donkey milk lactoferrin investigated by high-resolution mass spectrometry. Amino Acids 48, 2799–2808 (2016). https://doi.org/10.1007/s00726-016-2315-z

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