Comprehensive profiling and development of a collision cross section database for milk oligosaccharides via orthogonal UPLC-cyclic ion mobility-mass spectrometry system

Human milk oligosaccharides (HMOs) have attracted immense interest in the infant formula industry for their health benefits. Herein, we utilized liquid chromatography-cyclic ion mobility-mass spectrometry (LC-cIM-MS) to develop a robust and multidimensional HMO profiling workflow. This workflow relies on a self-built glycan library, allowing high-throughput searching of oligosaccharides . cIM-MS demonstrated high resolving power in discriminating glycan isomers and increasing peak capacity. This also facilitated the accurate elucidation of most oligosaccharides at sequence levels. A remarkably diverse milk oligosaccharide profile ( n = 98) was observed and enabled the discovery of distinctive chromatographic retention patterns. To provide supplementary selectivity for future routine assignment in the absence of standards, we further developed a comprehensive database of experiment-derived traveling wave collision cross section in nitrogen ( TW CCS N2 ) for 98 HMOs, including isomer-resolved TW CCS N2 values. Finally, the profile revealed 64 oligosaccharides unique to human milk compared with infant formula, indicating the potential ingredients for formula improvement. • A multidimensional profiling workflow for HMOs based on LC-cIM-MS was developed. • cIM-MS remarkably improved the resolution of HMO isomers and peak capacity. • Clear MS/MS spectra enable in-depth elucidation of HMOs at sequence level. • A robust isomer-resolved TW CCS database was developed for HMO routine assignment. • 64 HMOs were found unique to human milk compared with infant formula.