Amino acid sequence and glycan compositions of donkey s milk lactoferrin by 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, 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 s lactoferrin sequence, that, at least for the covered sequence, differs from the mare s 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). Glycosylation, one of the most common post-translational modifications, can modify the structural conformation of the protein and consequently its biological activity. A comprehensive glycosylation profile by chymotrypsin digestion, TiO2 and HILIC enrichment strategy, reversed-phase high performance liquid chromatography, electrospray mass spectrometry, high collision dissociation fragmentation, is reported. 26 different glycan compositions were identified, linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 47

Zeus, Aesculapius, Amalthea and the proteome of goat milk

he 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. 2g for each different pH captures) vs. barely 100μL 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

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