Quantifying Protein-Carbohydrate Interactions Using Liquid Sample Desorption Electrospray Ionization Mass Spectrometry

The application of liquid sample desorption electrospray ionization mass spectrometry (liquid sample DESI-MS) for quantifying protein-carbohydrate interactions in vitro is described. Association constants for the interactions between lysozyme and beta-D-GlcNAc-(1 -> aEuro parts per thousand 4)-beta-D-GlcNAc-(1 -> aEuro parts per thousand 4)-D-GlcNAc and beta-D-GlcNAc-(1 -> aEuro parts per thousand 4)-beta-D-GlcNAc-(1 -> aEuro parts per thousand 4)-beta-D-GlcNAc-(1 -> aEuro parts per thousand 4)-D-GlcNAc, and between a single chain antibody and alpha-D-Galp-(1 -> aEuro parts per thousand 2)-[alpha-D-Abep-(1 -> aEuro parts per thousand 3)]-alpha-D-Manp-OCH3 and beta-D-Glcp-(1 -> aEuro parts per thousand 2)-[alpha-D-Abep-(1 -> aEuro parts per thousand 3)]-alpha-D-Manp-OCH3 measured using liquid sample DESI-MS were found to be in good agreement with values measured by isothermal titration calorimetry and the direct ESI-MS assay. The reference protein method, which was originally developed to correct ESI mass spectra for the occurrence of nonspecific ligand-protein binding, was shown to reliably correct liquid sample DESI mass spectra for nonspecific binding. The suitability of liquid sample DESI-MS for quantitative binding measurements carried out using solutions containing high concentrations of the nonvolatile biological buffer phosphate buffered saline (PBS) was also explored. Binding of lysozyme to beta-D-GlcNAc-(1 -> aEuro parts per thousand 4)-beta-D-GlcNAc-(1 -> aEuro parts per thousand 4)-D-GlcNAc in aqueous solutions containing up to 1x PBS was successfully monitored using liquid sample DESI-MS; with ESI-MS the binding measurements were limited to concentrations less than 0.02 X PBS.