Advances in analysis of human milk oligosaccharides

Abstract

Oligosaccharides in human milk strongly influence the composition of the gut microflora of neonates. Because it is now clear that the microflora play important roles in the development of the infant immune system, human milk oligosaccharides (HMO) are studied frequently. Milk samples contain complex mixtures of HMO, usually comprising several isomeric structures that can be either linear or branched. Traditionally, HMO profiling was performed using HPLC with fluorescence or UV detection. By using porous graphitic carbon liquid chromatography MS, it is now possible to separate and identify most of the isomers, facilitating linkage-specific analysis. Matrix-assisted laser desorption ionization time-of-flight analysis allows fast profiling, but does not allow isomer separation. Novel MS fragmentation techniques have facilitated structural characterization of HMO that are present at lower concentrations. These techniques now facilitate more accurate studies of HMO consumption as well as Lewis blood group determinations.

Figure 1

Separation of isomers of reduced FS-LNH using nanoliquid chromatography porous graphitic carbon chip time-of-flight MS. RT, retention time; FS-LNH, fucosyl-lacto-N-hexaose. Reprinted from Reference (7) with permission.

Figure 2

Separation of 8-aminopyrene-1,3,6-trisulfonic acid–labeled human milk oligosaccharides using capillary electrophoresis with laser-induced fluorescence (LIF) (A) and capillary electrophoresis with LIF MS (B). The adjustments needed for hyphenation with MS result in reduced resolving power and longer analysis times. MS, however, facilitates direct identification. Reprinted from Reference (47) with permission.

Figure 3

Matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance MS spectrum of reduced human milk oligosaccharides (HMO) using 2,5-dihydroxybenzoic acid matrix in the positive ionization mode. Signals originating from HMO are marked with a diamond. Reprinted from Reference (52) with permission.

Figure 4

Differentiation of 3 fucosylated human milk oligosaccharides (HMO) using collision-induced dissociation fragmentation of deprotonated species in the negative ionization mode. MS³ fragmentation spectra of m/z 1079 for the 3 fucosylated HMO (A–C) are depicted together with fragmentation patterns explaining the most important ions. Reprinted from Reference (56) with permission.