Preparative scale release and purification of N-linked glycan standards from human milk glycoproteins

Glycosylation has been implicated in many important biological processes, disease states, and host-pathogen interactions yet progress in understanding the biological roles and functions of glycosylation has been limited by the lack of standardised tools and techniques. Purified glycans of specific structure are extremely valuable as standards for structural characterisation of glycans as well as for the calibration of instruments, and the validation or comparison of analytical techniques. However, structurally defined glycans have proven difficult to obtain in ample quantities, are extremely complex to produce synthetically, and when commercially available, are considerably expensive. In this work, a method was developed for obtaining preparative scale quantities of specific N-linked glycan structures of natural origin in a laboratory based setting, as a means to prepare glycan standards for analysis and other downstream processes. Human milk was selected as a biologically relevant source of N-linked glycans due to the high glycoprotein content and growing interest in the activity of human milk glycoconjugates. A protocol for the preparative scale release of N-linked glycans from human milk proteins and a separation technique using high performance liquid chromatography (HPLC) on porous graphitised carbon (PGC) was developed. A method of offline detection was also established using matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to identify N-linked glycans in the fractions separated by PGC-HPLC. Six structurally unique N-linked glycans from human milk were separated and collected at high purity (~90%). Characterisation by PGC-LC coupled to electrospray ionisation tandem mass spectrometry (PGC-LC-ESI-MS/MS) identified a set of fucosylated N-linked glycans, structures which are not currently commercially available. Fucosylation is a specific feature of human glycosylation that is of recent interest to the medical research field, hence the development of a preparative scale technique to obtain fucosylated glycans is of significant value. A retention time library and MS fragmentation dataset of fucosylated glycans was established to facilitate their use as standards. In addition, the application of the human milk N-linked glycans in an in vitro assay was investigated, in which the glycans were assessed for their potential to inhibit bacterial binding to membrane proteins extracted from human intestinal cells.