Mass Spectrometric Analysis of Human Milk Oligosaccharides: Correlating Glycan Expression to Maternal Phenotypes and Infant Healt

Human milk is a multifaceted biofluid that not only provides nourishment for neonates, but also contributes to their protection from pathogenic infections. Human milk oligosaccharides (HMOs) are an abundant, bioactive component of breast milk, and serve a variety of biological functions. Among the most studied are their prebiotic and antipathogenic properties, by which HMOs protect the infant from pathogens by selectively promoting the growth of commensal bacteria, as well as behaving as receptor analogs to impede the binding of harmful pathogens to cell surfaces. To better understand the biological role of HMOs, sensitive and in-depth analyses of the milk glycome are required. Presented in this dissertation is the development of a rapid-throughput, mass spectrometry-based method for profiling the oligosaccharides in human milk. The methods described herein incorporate the use of nano-scale liquid chromatography performed on a microfluidic chip, coupled to time-of-flight mass spectrometry (nanoLC Chip-TOF MS) to generate comprehensive glycoprofiles of human milk. With the development of a 96-well plate-based platform for sample preparation, and the application of an annotated milk oligosaccharide library, rapid structure identification in large-scale cohorts is made possible. This dissertation explores the maternal characteristics and phenotypes that effect glycan expression during lactation in women from populations around the world. These rapid-throughput methods are also used to profile the in vitro consumption of HMOs by various species of Bifidobacterium that often dominate the intestinal microbiota of breastfed infants. Furthermore, the initiation of a fecal glycan library is described, providing evidence for the presence of intact HMOs and N-glycans, as well as their digestion products, in infant stool. Comprehensively profiling the fecal glycome provides means for characterizing the in vivo digestion of milk glycans.