Sunday, May 11, 2025
5/11/2025
A bioanalytical research program to unravel the human milk glycome - PROJECT SUMMARY While human milk oligosaccharides (HMOs) are involved in a variety of biological processes that enable the healthy development of the brain-gut axis of infants, a true understanding of how their molecular structures and composition dictate such functions has largely remained a mystery. HMOs possess a tremendous degree of both structural complexity and isomeric heterogeneity due to their many possible permutations of linkage positioning and monosaccharide constituent arrangements. Therefore, improved analytical advancements are necessary to unravel this isomerism puzzle and gain a better understanding of their infant-specific roles. Mass spectrometry (MS) in conjunction with liquid chromatographic (LC) separations has been broadly used for HMO analyses. Unfortunately, in many instances, LC-MS-based techniques suffer from insufficient chromatographic resolution resulting in convoluted MS/MS spectra for co-eluting species, and thus limiting the accurate identification and structural elucidation of HMOs. The overall goal of our research program is to develop a bioanalytical workflow to enable more effective characterization of HMOs. To achieve this, we will implement a multidimensional separations platform using recycling liquid chromatography coupled to high- resolution cyclic ion mobility separations and mass spectrometry in conjunction with the development of solution and gas-phase chemical probes and top-down sequencing approaches. Our multidimensional separations platform will not only enable improved peak capacity, but also higher resolution separations to facilitate the characterization of previously indistinguishable HMO isomers. Additionally, our cyclic ion mobility- based separations will allow the determination of highly precise collision cross section values that will be disseminated to other laboratories in the glycobiology community. Our chemical probes will facilitate the improved identification of glycosidic linkage positioning in isomeric HMOs, especially when no authentic standard is present. Lastly, our top-down sequencing strategies will enable the arrangement of monosaccharide constituents to be better determined. Overall, we envision that our proposed bioanalytical toolbox will make strides toward the de novo sequencing of HMOs and help achieve our overarching goal of unraveling the human milk glycome to identify essential HMOs to be incorporated into infant formula.
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