Targeted LC-ESI-MS2 characterization of human milk oligosaccharide diversity at 6 to 16\xa0weeks post-partum reveals clear staging effects and distinctive milk groups

Marko Mank,Bernd Stahl,Albert J R Heck,Hans Hauner

doi:10.1007/s00216-020-02819-x

Abstract

Many molecular components in human milk (HM), such as human milk oligosaccharides (HMOs), assist in the healthy development of infants. It has been hypothesized that the functional benefits of HM may be highly dependent on the abundance and individual fine structures of contained HMOs and that distinctive HM groups can be defined by their HMO profiles. However, the structural diversity and abundances of individual HMOs may also vary between milk donors and at different stages of lactations. Improvements in efficiency and selectivity of quantitative HMO analysis are essential to further expand our understanding about the impact of HMO variations on healthy early life development. Hence, we applied here a targeted, highly selective, and semi-quantitative LC-ESI-MS2 approach by analyzing 2 × 30 mature human milk samples collected at 6 and 16 weeks post-partum. The analytical approach covered the most abundant HMOs up to hexasaccharides and, for the first time, also assigned blood group A and B tetrasaccharides. Principal component analysis (PCA) was employed and allowed for automatic grouping and assignment of human milk samples to four human milk groups which are related to the maternal Secretor (Se) and Lewis (Le) genotypes. We found that HMO diversity varied significantly between these four HM groups. Variations were driven by HMOs being either dependent or independent of maternal genetic Se and Le status. We found preliminary evidence for an additional HM subgroup within the Se- and Le-positive HM group I. Furthermore, the abundances of 6 distinct HMO structures (including 6′-SL and 3-FL) changed significantly with progression of lactation.Graphical abstract

Highlights

Human milk (HM) is largely defined by the abundance of 5 major constituents comprising lactose, lipids, oligosaccharides, proteins, and water
Our multiple reaction monitoring (MRM) LC-ESI-MS2 approach with enhanced specificity for fucose positional human milk oligosaccharides (HMOs) isomers was applied to 60 unknown human milk samples
These major HMO structures cover approximately > 50% of all HMOs contained in HM by quantity

Summary

Introduction

Human milk (HM) is largely defined by the abundance of 5 major constituents comprising lactose, lipids, oligosaccharides, proteins, and water. Pre-treatment of human milk samples, targeted semi-quantitative negative ion MRM LC-ESI-MS2 analyses of HMOs, software settings, evaluation of data, and manual assignment of human milk groups were essentially conducted as recently published in more detail by Mank et al [73]. The correct identification of the latter HMO structures via their respective MRM transitions was proven by analysis of pure HMO standards (concentrations ~ 0.03 to 0.05 mg/ml) which were run together with the INFAT human milk study samples. To investigate if abundances of individual HMO structures differed significantly between human milk groups as identified by PCA, non-parametric Kruskal-Wallis H testing [77] was performed with SPSS statistics software version 19.0.0 (IBM, Armonk, USA). Results were displayed as bar charts created with Microsoft Excel for Office 365

Results and discussion

18 Total SL

Conclusions

Compliance with ethical standards