Robust and High-Resolution All-Ion Fragmentation LC-ESI-IM-MS Analysis for In-Depth Characterization or Profiling of Up to 200 Human Milk Oligosaccharides

Human milk oligosaccharides (HMOs) represent the third most abundant fraction of biomolecules in human milk (HM) and play a crucial role in infant health and development. The unique contributions of HMOs to healthy development of breast-fed infants are assumed to rely on the extraordinary complexity and diversity of HMO isomeric structures, which in turn still cause a huge analytical challenge. Many contemporary analytical methods aiming for more detailed HMO characterization combine ion mobility (IM) with LC-MS for enhanced structural resolution but are typically lacking the robustness necessary for application to HM cohorts with hundreds of samples. To overcome these challenges, we introduce a novel, robust all-ion fragmentation (AIF) LC-ESI-IM-MS method integrating four analytical dimensions: high-resolution LC separation, IM drift time, accurate mass precursor, and fragment ion measurements. This four-dimensional (4D) analytical characterization is sufficient for resolving various HMO structural isomers in an efficient way. Thereby, up to 200 HMO compounds with a maximum degree of polymerization of 13 could be simultaneously identified and relatively quantified. We devised two methods using this 4D analytical approach. One intended for in-depth characterization of multiple known but also novel HMO structures and the second is designed for robust, increased-throughput analyses. With the first approach, five trifucosyl-lacto-N-tetraose isomers (TF-LNTs), four of which were never detected before in HM, as well as additional difucosyl-lacto-N-heaose isomers (DF-LNHs), were revealed and structures fully elucidated by AIF and IM. This exemplifies the potential of our method for in-depth characterization of novel complex HMO structures. Furthermore, the increased-throughput method featuring a shorter LC gradient was applied to real-world HM samples. Here, we could differentiate the HM types I-IV based on a broader range of partly new marker HMOs. We could also derive valuable new insights into variations of multiple and rare HMOs up to DP 11 across lactational stages. Overall, our AIF LC-ESI-IM-MS approach facilitates in-depth monitoring and confident identification of a broad array of distinct and simple to very complex HMOs. We envision this robust AIF LC-ESI-IM-MS approach to advance HMO research by facilitating the characterization of a broad range of HMOs in high numbers of HM samples. This may help to further extend our understanding about HMOs structure-function relationships relevant for infants' healthy development.