Microfluidics-Mass Spectrometry of Protein-Carbohydrate Interactions: Applications to the Development of Therapeutics and Biomarker Discovery.

Abstract

The functional interactions of carbohydrates and their protein receptors are the basis of biological events critical to the evolution of pathological states. Hence, for the past years, such interactions have become the focus of research for the development of therapeutics and discovery of novel glycan biomarkers based on their binding affinity. Due to the high sensitivity, throughput, reproducibility, and capability to ionize minor species in heterogeneous mixtures, microfluidics-mass spectrometry (MS) has recently emerged as a method of choice in protein-glycan interactomics. In this chapter, a straightforward microfluidics-based MS methodology for the assessment of protein-glycan interactions is presented. The general protocol encompasses: (1) submission of the interacting partners to a binding assay under conditions mimicking the in vivo environment; and (2) screening of the reaction products and their structural characterization by fully automated chip-nanoelectrospray (nanoESI) MS and multistage MS. The first section of the chapter is devoted to describing a method that enables the study of protein-oligosaccharide interactions by chip-nanoESI quadrupole time-of-flight (QTOF) MS and top-down complex analysis by collision-induced dissociation (CID). This section provides the protocol for the determination of the complex formed by standard β-lactoglobulin (BLG) with maltohexaose (Glc6) and recommends as a concrete application the study of the interaction between BLG extracted from human milk with Glc6, considered a ligand able to reduce the allergenicity of this protein. The second part is dedicated to presenting the protocols for the binding assay followed by chip-nanoESI ion trap (ITMS) and electron transfer dissociation (ETD) in combination with CID for protein-ganglioside interactions, using as an example the B subunit of cholera toxin (Ctb5) in interaction with comercially available GM1 species. The methodology described may be successfully applied to native ganglioside mixtures from human brain, in particular for discovery of biomarkers on the basis of their binding affinity.