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-