Lewis Blood Group Detection from Human Milk Oligosaccharide Mass Finger Prints

The structural diversity of human milk oligosaccharides (HMOs) strongly corresponds with the Lewis (Le) blood group status of the individual donor. The three different Lewis blood groups, i.e., Le(a-b+), Le(a+b-) and Le(a-b-) exhibit different expression levels of fucosyltransferases which result in a great structural variety, in particular, in the case of neutral fucosylated HMO species. These differences in the oligosaccharide patterns are the basis for our mass spectrometric approach which allows us to assign a milk sample to one of the three groups. Starting from fifty microliters of human milk the established method provides a time and material saving way for a Lewis blood group classification of milk samples. The relative abundance of diagnostically relevant compositional species, such as, Hex₂Fuc₂, Hex₃HexNAc₁Fuc₂ and Hex₄HexNAc₂Fuc₃ in MS profile spectra is used as a first step for the identification. In a second step MS/MS analyses of characteristic precursor ions are performed. For each Lewis blood group, specific mass profiles and fragment ion patterns could be identified allowing a rapid classification without the need of a blood sample. Furthermore, the outlined protocol can be used for rapid screening in clinical studies to gain detailed information about neutral as well as acidic oligosaccharide patterns of specific samples, allowing also a relative quantification of individual compositional glycan species. Moreover, the described analytical approach enables an easy quality control of milk samples acquired from milk banks.