Advances in Native Mass Spectrometry Tools to Quantify Immune Lectin-Glycan Interactions
Creators: Bui, Duong T
Publisher: name: University of Alberta Library
Year: 2024

<p>Glycans (oligosaccharides) are an important class of biomolecules essential to all living organisms. All cells are coated with a dense layer of glycoconjugates (glycan bearing molecules), such as glycoproteins, glycolipids, glycoRNA, which is called the glycocalyx. Through their interactions with glycan-binding proteins (GBPs), glycans participate in many physiological and pathophysiological processes. As part of the immune system, glycans interact with immune lectins to regulate the innate immune response and homeostasis of adaptive immune response, and are involved in tumor immunity, inflammation, and autoimmunity. As glycan-GBP interactions shape our immunity, uncovering the natural glycan ligands of the immune lectins is critical to the development of diagnostics and therapeutics targeting the immune systems. There are several outstanding analytical challenges that have hindered the characterization of the natural ligands of most immune lectins. These include the large number of possible glycan structures and their varied presentations, the effect of the environment on their binding properties, and the low affinities of their monovalent interactions, typically at or below the detection limit of standard binding assays. There exist several analytical techniques that are currently used for detection and quantification of glycan-GBP interactions, each with particular strengths and weaknesses. Recently, native mass spectrometry (nMS), which uses of electrospray ionization (ESI) performed under native-like solution conditions, has emerged as a highly versatile, label- and immobilization-free tool for the discovery of glycan ligands of GBPs and quantification of their stoichiometry and thermodynamic parameters. However, as with all analytical techniques, there are challenges that hinder the full potential of nMS to be realized. This dissertation addresses several long-standing challenges in nMS-based discovery and quanitification of glycan-GBP interactions. <br>
    Slow mixing mode (SLOMO) - nMS was developed to overcome a significant barrier – non-uniform ESI-MS response factors – to quantify lectin-glycoconjugate interactions in vitro. The technique exploits the slow diffusion of analyte in the confined space of a nanoflow ESI tip to directly measure relative response factors of interacting species and, simultaneously, determine affinity. This technological breakthrough is critical to efforts to quantify immune lectin binding to glycoproteins and to glycolipids presented in model membranes. Moreover, SLOMO-nMS is a general method and can be applied to a wide range of biomolecular complexes. While the SLOMO-nMS technique was originally demonstrated for 1:1 interactions, subsequent efforts demonstrated its suitability for quantifying coupled equilibria, thereby extending the utility of this method.<br>
    Mass spectrometry-based shotgun glycomics (MS-SG) is a high-throughput method for the discovery of natural ligands of lectins. MS-SG was developed to address the limited availability of glycans in purified form. To implement MS-SG, natural libraries of glycans derived from glycoconjugates in cells or tissue are screened against a target lectin using catch-and-release nMS. Because glycan concentrations are unknown, the affinities of ligands detected in natural libraries cannot be determined. To overcome this challenge, the implementation of SG-MS using fluorophore-labeled libraries, which can be quantified using liquid chromatography, to both detect and rank ligand affinities was explored. This method was tested using a series of sialic acid-binding lectins and natural N-glycan libraries. While easily implementable, the affinity rankings obtained with this approach are sensitive to the nature of the fluorophore added. Recognizing the need to eliminate undesirable effects of glycan labeling, concentration-independent (COIN)-nMS was developed. This label-free approach, which can be applied to mixtures of glycans at unknown concentrations, enables the affinities of all detected interactions to be measured simultaneously. In addition, this method can be applied to quantify interactions of heterogenous GBPs, which represents a significant challenge for conventional nMS binding measurements. The results of quantitative screening of natural N-glycan and glycopeptide libraries against human, bacterial, viral and immune lectins serve to illustrate the power of COIN-nMS for mapping glycan interactions of endogenous and exogenous lectins important to human health and disease.</p>