Jasmine C. C. Davis; Sarah M. Totten; Carlito B. Lebrilla
UC Davis, Davis, CA
This research aimed to create a library of intact and degraded milk glycans found in infant feces from microbial catabolism.
Oligosaccharides continue to be one of the most difficult biomolecules to analyze. Analysis is, however, aided by the use of biological rules followed by glycosyl transferases and glycosidases in their modification of the nascent glycan structure. Human milk oligosaccharides (HMOs) will interact with bacteria in the infant’s digestive tract, but the nature of these interactions is often unknown. Furthermore, the interactions often lead to the production of new types of oligosaccharides that are degradation products from the bacterial consumption. In this this research, we developed a method for determining glycan products due to the interactions of bacteria with glycoconjugates in milk. We are able to rapidly identify glycan structures and correlate the enzymes responsible for those oligosaccharide products.
Fecal samples were diluted and shaken overnight. Proteins in the supernatant were precipitated with ethanol, and the resulting glycans were reduced to their alditol form with NaBH4. The samples were cleaned up with solid phase extraction, the eluents evaporated, and before analysis the samples were reconstituted in water.
Extracted glycans were analyzed on a nano-HPLC-Chip/TOF MS system followed by identification and quantitation with Agilent Mass Hunter Qualitative Analysis. Structures were confirmed using collision induced dissociation (CID) on a nano-HPLC-Chip/Q-TOF MS.
To monitor bacterial consumption, an HMO pool was digested with a ß-galactosidase, incubated, and C18 zip-tip cleaned up. Spectra of the consumed HMOs were compared to that of the undigested HMO pool, and CID was used for structural confirmation.
Consumption studies were performed to determine the degradation products of a ß-galactosidase strain, as well as the specificity of that strain. Our intact HMO library contains specific linkages of isomers, so we are able to determine the specificity of the enzyme by comparing the spectra and abundances of isomers from an undigested HMO pool with the consumed pool. The masses and compositions of the digested products were added to a previous HMO library in order to create a fecal library.
Not only are there exoglycosidases that cleave glycans, but there are also enzymes that can cleave N-glycans from glycoproteins. There are no free N-glycans in breast milk, but they were discovered in the fecal samples analyzed, so those structures, along with their degraded products, were also taken into account. Compositions of intact N-glycans from a theoretical library were added to the fecal library.
Structures of the extracted glycans, both intact and degraded, were confirmed using CID. The CID spectra of the N-glycans were not consistent with the N-glycans containing their intact chitobiose core, and it appeared that the N-acetylglucosamines (GlcNAcs) on their reducing ends were missing from their structures, suggesting the glycan was cleaved by an endoglycosidase. The possible digested N-glycans were also added to the fecal library to incorporate all possible glycan compositions.
Our library was created with specific isomer information, with identification based on composition, exact mass, and retention time. We can monitor the identified degradation products in fecal samples based on the data from the consumption studies. It is possible to tell the difference between most HMOs and N-glycans based on their differences in composition and retention times. HMOs and N-glycans both have specific biological rules in how glycosyl transferases add monosaccharides to their core structures, so there are certain compositions that either glycan can have.