Coupling Mass Spectrometry-Based "Omic" Sciences with Bioguided Processing to Unravel Milk's Hidden Bioactivities

Abstract

Many of milk's functional molecules could not be discovered until the right concordance of novel separation and analytical technologies were developed and applied. Many health-promoting components still await discovery due to technical challenges in their identification, isolation and testing. As new analytical technologies are assembled, new functional milk molecules will be discovered. Bovine milk is a source of a wide array of known bioactive compounds from a variety of molecular classes, including free glycans, lipids, glycolipids, peptides, proteins, glycoproteins, stem cells and microRNA. Because milk is such a complex mixture, when analyzed without fractionation or purification, many components mask the analytical signal of others, so some components cannot be detected. Modern analytics allow for the discovery and characterization of hundreds of novel milk compounds with high-resolution and high-accuracy. Liquid chromatography paired with electrospray ionization allows the separation of peptides, glycans and glycolipids for improved mass spectrometric detection. Target proteins and glycoproteins can now be purified from intact milk or other dairy streams by chromatography in order to better characterize these proteins for new bioactivities. The combination of advanced analytics with the new engineering capabilities will allow for high molecular resolution and separation techniques that can be scaled-up to semi-industrial and industrial scale for translation of lab-based discoveries. Bioguided analysis and design of dairy processing side streams will result in the transformation of waste into isolated functional ingredients to add value to dietary products.

Keywords: Bioactive Molecules; Bioguided Processing; Glycan; Glycolipid; Glycoprotein; Mass Spectrometry; Milk; Oligosaccharide; Peptide; Purification.

Figure 1

Basic components of mass spectrometers.

Figure 2

Representative MALDI-MS spectra of gangliosides from bovine and human milk. Negative mode MALDI MS spectrum of (A) bovine milk gangliosides and (B) human milk gangliosides. The inserts show the structures of GM3 and GD3 gangliosides.

Figure 3

Example bioinformatic library for fucosylated BMO. Each entry includes retention time (RT), accurate mass, oligosaccharide composition (Hex (hexose-galactose in yellow and glucose in blue circles), HexNAc (N-acetylhexosamine in white squares) Fuc (fucose-red triangles), NeuAc, and NeuGc (N-glycolylneuraminic acid) and full/partial structure.

Figure 4

Schematic of enzymatic N-glycan release from protein and workflow for characterization of the released glycans.

Figure 5

Electrophoretic gels demonstrate the sequential successful purification of target whey proteins (e.g. lactoferrin from the remaining whey proteins). Isolated fractions were analyzed on 12% SDS-PAGE after affinity chromatography.

Figure 6

Schematic of potential pathways to maximize the recovery of milk bioactive components.