Human milk components have a wide range of biological functions that contribute to the growth, immune development and long-term health of newborns. Free Oligosaccharides are the third most abundant solid component in human milk. They demonstrate a number of functions including serving as prebiotics to stimulate the growth of beneficial intestinal bacteria, as receptor analogs to inhibit binding of pathogens, and as substances that promote postnatal brain development. To better analyze oligosaccharide structures will help researchers understand their biological roles. A well-developed human milk glycome will give insight into the structure-function relation. This dissertation demonstrates mass spectrometry based methods to provide a systematic and comprehensive study of HMO structures. The methods incorporate high resolution FT-ICR MS, Chip-based nano-LC/MS, and program assisted high-throughput structural identification. Monosaccharide composition can be easily investigated by MALDI FT-ICR MS. IRMPD generates informative fragment ions for structural elucidation. HPLC-Chip/TOF MS provides a sensitive and quantitative method for sample profiling. A microchip-based nano-LC column packed with porous graphitized carbon (PGC) provides excellent isomer separation for oligosaccharides. By applying proper collision energy, HPLC-Chip/Q-TOF MS further differentiate isomers with distinct tandem MS spectra. Together with exoglycosidase digestion, the linkages including epitopes such as Lewis type were elucidated. In order to facilitate the structure identification for other milk samples, retention times, accurate masses and MS/MS spectra of over 70 structures were deduced and incorporated into the Agilent Personal Compound Database and Library software to generate a library file. Automatic fragment peak search between sample data and library spectra was performed using MassHunter Qualitative Analysis program. Oligosaccharide structures in human milk samples can be rapidly and accurately identified. This structure library was further applied to identify primate milk oligosaccharide structures. The result shows intriguing relation between human and animal species linked through evolution. This method can also be applied to other type of glycans.
|Advisor:||Lebrilla, Carlito B.|
|Commitee:||German, J. Bruce, Land, Donald P.|
|School:||University of California, Davis|
|School Location:||United States -- California|
|Source:||DAI-B 73/10(E), Dissertation Abstracts International|
|Subjects:||Analytical chemistry, Biochemistry|
|Keywords:||Human milk oligosaccharides, Mass spectrometry, Primate milk oligosaccharides, Structure library, Tandem ms|
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