Rapid structural characterization of human milk oligosaccharides and distinction of their isomers using trapped ion mobility spectrometry time-of-flight mass spectrometry.

Oligosaccharides have multiple functions essential for health. Derived from the condensation of two to several monosaccharides, they are structurally diverse with many co-occurring structural isomer families, which make their characterization difficult. Thanks to its ability to separate small molecules based on their mass, size, shape, and charge, ion mobility-mass spectrometry (IM-MS) has emerged as a powerful tool for separating glycan isomers. Here, the potential of such a technique for the rapid characterization of main human milk oligosaccharides (HMOs) was investigated. Our study focused on 18 HMO standards. The IM-MS analysis enabled to distinguish almost all the HMOs studied, in particular thanks to the single ion mobility monitoring acquisition using the trapped ion mobility spectrometry device, providing high ion mobility resolution and enhanced ion mobility separation. Alternatively, the combination of IM-MS separation with MS/MS experiments has proven to increase performance in identifying HMOs and especially isomers poorly separated by ion mobility alone. Finally, collision cross-section values are provided for each species generated from the 18 HMOs standards, which can serve as an additional identifier to characterize HMOs.