Protein glycosylation is an important post-translational modification. It is a feature that enhances the functional diversity of proteins and influences their biological activity. A wide range of functions for glycans have been described, from structural roles to participation in molecular trafficking, self-recognition and clearance. Understanding the basis of these functions is challenging because the biosynthetic machinery that constructs glycans executes sequential and competitive steps that result in a mixture of glycosylated variants (glycoforms) for each glycoprotein. Additionally, naturally occurring glycoproteins are often present at low levels, putting pressure on the sensitivity of the analytical technologies. No universal method for the rapid and reliable identification of glycan structure is currently available; hence, research goals must dictate the best method or combination of methods. To this end, we introduce some of the major technologies routinely used for structural N- and O-glycan analysis, describing the complementary information that each provides.

The present study was conducted to obtain a comprehensive overview of oligosaccharides present in the milk of a variety of important domestic animals including cows, goats, sheep, pigs, horses and dromedary camels. Using an analytical workflow that included ultraperformance liquid chromatography -hydrophilic interaction liquid chromatography with fluorescence detection coupled to quadrupole time-of-flight MS, detailed oligosaccharide libraries were established. The partial or full characterisation of the neutral/fucosylated, phosphorylated and sialylated structures was facilitated by sequencing with linkage-and sugar-specific exoglycosidases. Relative peak quantification of the 2-aminobenzamide-labelled oligosaccharides provided additional information. Milk from domestic animals contained a much larger variety of complex oligosaccharides than was previously assumed, and thirteen of these structures have been identified previously in human milk. The direct comparison of the oligosaccharide mixtures reflects their role in the postnatal maturation of different types of gastrointestinal systems, which, in this way, are prepared for certain post-weaning diets. The potential value of animal milk for the commercial extraction of oligosaccharides to be used in human and animal health is highlighted.Key words: Domestic animal milk: Oligosaccharides: Ultra-performance liquid chromatography -hydrophilic interaction liquid chromatography with fluorescence detection -MS: 2-Aminobenzamide: Relative quantification Human milk and animal milk are rich sources of bioactive oligosaccharides, which are of great interest to the functional food industry. Many biological activities have been reported for certain milk oligosaccharides including prebiotic activity, anti-adhesion effects, anti-inflammatory properties, glycomemodifying activity, and a role in brain development and growth-related characteristics of intestinal cells (1 -4) . Milk oligosaccharides are typically composed of three to ten monosaccharide units, including glucose (Glc), galactose (Gal) and N-acetyl-glucosamine (GlcNAc) as well as fucose and sialic acids. The core unit present at the reducing end of milk oligosaccharides is either lactose (Gal(b1 -4)Glc) or N-acetyl-lactosamine (Gal(b1 -4)GlcNAc) (5) .As the biological activity of milk oligosaccharides is dependent on their individual structural characteristics, a detailed knowledge of their composition is necessary. Human milk is a rich source of oligosaccharides (20 mg/l in colostrum; 12 -13 mg/l in mature milk), and more than 200 structures have been identified so far (6,7) . Less is known about animal milk oligosaccharides as their concentration in milk is low. For example, the concentration of oligosaccharides in bovine colostrum is 1 g/l, whereas their concentration in mature milk is about 20-fold less (8,9) . Using a combination of advanced analytical techniques, structural libraries of up to forty and even more than fifty bovine milk oligosaccharides (10 -12) , twenty-nine porcine milk oligosacch...

Although the properties of milk oligosaccharides have been of scientific interest for many years, their structural diversity presents a challenging analytical task. In the quest for a simple and robust technology to characterize the different oligosaccharides present in milk, we developed an analytical scheme based on their fluorescent labeling, pre-fractionation by weak anionic exchange chromatography and separation by hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC). HILIC relies on the hydrophilic potential of the molecule, which accounts for differences in properties such as molecular volume, lipophilic surface area, charge, composition, structure, linkage and oligosaccharide branching. The robustness of the methodology has been demonstrated using bovine colostrum oligosaccharides as a case study. Structural assignments for 37 free glycans, including 20 sialylated species, were obtained by a combination of HILIC-HPLC, exoglycosidase digestion and offline negative-ion mode mass spectrometry (MS)/MS. Parameters obtained for each glycan, including linkages, enzymatic digestion products and glucose unit values, will be added to GlycoBase, a public access database (http://glycobase.nibrt.ie/glycobase.html). This approach provides a basis for the analysis of free milk oligosaccharides in a fast and sensitive manner and could be adapted for an automated technology platform amenable to diverse environments. Indeed, our approach, in conjunction with bacterial-binding assays, can provide a better understanding of the structural elements required for biological activity of free milk oligosaccharides and could serve as a scientific basis for the selection of such bioactives from various food sources.