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

Abstract

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 β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-D-GlcNAc and β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-D-GlcNAc, and between a single chain antibody and α-D-Galp-(1 → 2)-[α-D-Abep-(1 → 3)]-α-D-Manp-OCH3 and β-D-Glcp-(1 → 2)-[α-D-Abep-(1 → 3)]-α-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 β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-D-GlcNAc in aqueous solutions containing up to 1× 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.

Influence of Sulfolane on ESI-MS Measurements of Protein–Ligand Affinities

“…This value is in reasonable agreement with previously reported values, 0.8-1.1 × 10 5 M -1 [34,41,42]. Upon addition of 2% sulfolane to the solution, the charge states of the Lyz n+ and (Lyz + L1) n+ ions shifted to n = 8-12 ( Figure 1b).…”

“…Binding measurements were carried out for aqueous solutions of Lyz (0.2 mM) and L1 (2.0 mM) with 2%, 5%, and 10% sulfolane, respectively ( Figures S1, S2, and S3, Supplementary Data). Plotted in Figure 3 are the corresponding K a values as well as the reported value measured in the absence of sulfolane [34], together with the values measured by ESI-MS. Notably, the ITC data reveal that sulfolane reduces the thermodynamic stability of the Lyz-L1 interaction in solution.…”

“…For the liquid sample DESI-MS measurements, a modified OMNI SPRAY ion source 2-D OS-6205 (Prosolia Inc., Indianapolis, IN, USA) was used. Details of the experimental setup can be found elsewhere [34]. Briefly, the liquid sample solution was delivered through a silica capillary (360 nm o.d., 100 nm i.d.)…”

“…The general procedure for determining association constants (K a ) for protein-ligand interactions from ESI mass spectra has been described in detail elsewhere [34,[36][37][38] and only a brief description is given for the case where the protein has a single ligand binding site. The assay relies on the detection and quantification of the gas-phase ions of the free and ligandbound protein.…”

“…Recently, it was shown that liquid sample and reactive liquid sample DESI-MS can be used to quantify protein--ligand affinities in solution [31,34]. An attractive feature of the liquid sample DESI-MS assay is that it is significantly more tolerant to nonvolatile buffers, such as PBS, than ESI-MS [31,34].…”