Proteins and bioactive peptides from donkey milk: The molecular basis for its reduced allergenic properties.

The legendary therapeutics properties of donkey milk have recently been supported by many clinical trials who have clearly demonstrated that, even if with adequate lipid integration, it may represent a valid natural substitute of cow milk for feeding allergic children. During the last decade many investigations by MS-based methods have been performed in order to obtain a better knowledge of donkey milk proteins. The knowledge about the primary structure of donkey milk proteins now may provide the basis for a more accurate comprehension of its potential benefits for human nutrition. In this aspect, experimental data today available clearly demonstrate that donkey milk proteins (especially casein components) are more closely related with the human homologues rather than cow counterparts. Moreover, the low allergenic properties of donkey milk with respect to cow one seem to be related to the low total protein content, the low ratio of caseins to whey fraction, and finally to the presence in almost all bovine IgE-binding linear epitopes of multiple amino acid differences with respect to the corresponding regions of donkey milk counterparts.

Polyphemus, Odysseus and the ovine milk proteome.

In the last years the amount of ovine milk production, mainly used to formulate a wide range of different and exclusive dairy products often categorized as gourmet food, has been progressively increasing. Taking also into account that sheep milk (SM) also appears to be potentially less allergenic than cow's one, an in-depth information about its protein composition is essential to improve the comprehension of its potential benefits for human consumption. The present work reports the results of an in-depth characterization of SM whey proteome, carried out by coupling the CPLL technology with SDS-PAGE and high resolution UPLC-nESI MS/MS analysis. This approach allowed the identification of 718 different protein components, 644 of which are from unique genes. Particularly, this identification has expanded literature data about sheep whey proteome by 193 novel proteins previously undetected, many of which are involved in the defence/immunity mechanisms or in the nutrient delivery system. A comparative analysis of SM proteome known to date with cow's milk proteome, evidenced that while about 29% of SM proteins are also present in CM, 71% of the identified components appear to be unique of SM proteome and include a heterogeneous group of components which seem to have health-promoting benefits. The data have been deposited to the ProteomeXchange with identifier .

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

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.

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

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 â†’ His), 328 (Thr â†’ Ile/Leu), 466 (Ala â†’ Gly), 642 (Asn â†’ Ser) and 668 (Ser â†’ 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.

Zeus, Aesculapius, Amalthea and the proteome of goat milk.

The goat whey proteome has been explored in depth via capture with combinatorial peptide ligand libraries (CPLLs) at three different pH values. A total of 452 unique species have been tabulated, a proteome discovery so far unmatched in any single other investigation of milk from any mammalian species. This massive discovery is probably related to: i) the extraordinary load of proteins onto the CPLL beads (i.e. 2 g for each different pH captures) vs. barely 100 µL of beads; ii) the high resolution/high mass accuracy of mass spectral data; and iii) the use of two complementary tools, Mascot and PEAKS, each one contributing to a set of unique protein IDs. Due to the relative paucity of available protein annotations for goat, only 10% of the identified proteins belong to the capra, whereas 52% are specific of sheep and 37% are homologous to that of bovine milk. This work reports the largest description so far of the goat milk proteome, which has been compared with cow's milk proteome and would thus help to understand the importance of low-abundance proteins with respect to the unique biological properties of this nutrient.