Amino Acids

, Volume 48, Issue 7, pp 1569–1580 | Cite as

Sequence characterization and glycosylation sites identification of donkey milk lactoferrin by multiple enzyme digestions and mass spectrometry

  • Serafina Gallina
  • Vincenzo Cunsolo
  • Rosaria Saletti
  • Vera Muccilli
  • Antonella Di Francesco
  • Salvatore FotiEmail author
  • Andrea Maria Lorenzten
  • Peter Roepstorff
Original Article


Lactoferrin, a protein showing an array of biochemical properties, including immuno-modulation, iron-binding ability, as well as antioxidant, antibacterial and antiviral activities, but which may also represent a potential milk allergen, was isolated from donkey milk by ion exchange chromatography. The characterization of its primary structure, by means of enzymatic digestions, SPITC derivatization of tryptic digest, reversed-phase high performance liquid chromatography, electrospray and matrix-assisted laser desorption/ionization mass spectrometry, is reported. Our results allowed the almost complete characterization of donkey lactoferrin sequence, that, at least for the covered sequence, differs from the horse genomic deduced sequence (UniProtKB Acc. Nr. O77811) by five point substitutions located at positions 91 (Arg → His), 328 (Thr → Ile/Leu), 466 (Ala → Gly), 642 (Asn → Ser) and 668 (Ser → Ala). Analysis of the glycosylated protein showed that glycans in donkey lactoferrin are linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476.


Lactoferrin Sequence characterization Glycosylation sites Donkey milk Mass spectrometry 



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. We are grateful to Dr D. Franchina and Dr K. Torrisi (ASILAT srl farm, Milo, Catania) for the gift of the raw donkey milk.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest with regards to publication of this research work.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study. Additional informed consent was obtained from all individual participants for whom identifying information is included in this article.


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Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  1. 1.Organic Mass Spectrometry Laboratory, Department of Chemical SciencesUniversity of CataniaCataniaItaly
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdenseDenmark

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