# Characterization of rare trifucosylated human milk oligosaccharides by cryogenic infrared ion spectroscopy (CIRIS)

## TL;DR

Scientists used a new technique to identify four rare sugar structures in human milk that were previously unknown.

## Contribution

A novel analytical method combining MS/MS and CIRIS was used to characterize previously ambiguous trifucosylated HMO isomers.

## Key findings

- Four novel isomers of trifucosylated oligosaccharides were identified in human milk.
- Some of these isomers could not be explained by known HMO biosynthetic pathways.
- CIRIS was used to determine the precise structures of isomeric HMO fragments.

## Abstract

While approximately 200 human milk oligosaccharide structures (HMOs) have been completely characterized, the precise isomeric structures of even more HMOs remain ambiguous. To address this analytical challenge, we employ tandem mass spectrometry (i.e., MS/MS) coupled with cryogenic infrared ion spectroscopy (CIRIS) to characterize and identify novel fucosylated HMOs isolated from pooled human milk. In this approach, the precise structure of isomeric precursor molecules purified by liquid chromatography (LC) is identified by IR fingerprinting their fragments. Using this technique, we have identified four novel isomers of trifucosylated oligosaccharides (i.e., trifucosyl-lacto-N-heptaoses and trifucosyl-lacto-N-neoheptaoses), some of which could not have been produced by previously known biosynthetic pathways for HMO synthesis in the mammary gland.

The online version contains supplementary material available at 10.1007/s00216-025-06114-5.