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.
Abstract. The results of an investigation into the influence of sulfolane, a commonly used supercharging agent, on electrospray ionization mass spectrometry (ESI-MS) measurements of protein-ligand affinities are described. Binding measurements carried out on four protein-carbohydrate complexes, lysozyme with β-D-GlcNAc-revealed that sulfolane generally reduces the apparent (as measured by ESI-MS) protein-ligand affinities. To establish the origin of this effect, a detailed study was undertaken using the lysozyme-tetrasaccharide interaction as a model system. Measurements carried out using isothermal titration calorimetry (ITC), circular dichroism, and nuclear magnetic resonance spectroscopies reveal that sulfolane reduces the binding affinity in solution but does not cause any significant change in the higher order structure of lysozyme or to the intermolecular interactions. These observations confirm that changes to the structure of lysozyme in bulk solution are not responsible for the supercharging effect induced by sulfolane. Moreover, the agreement between the ESI-MS and ITC-derived affinities indicates that there is no dissociation of the complex during ESI or in the gas phase (i.e., in-source dissociation). This finding suggests that supercharging of lysozyme by sulfolane is not related to protein unfolding during the ESI process. Binding measurements performed using liquid sample desorption ESI-MS revealed that protein supercharging with sulfolane can be achieved without a reduction in affinity.
Anisotropic particles have generated an enormous amount of research interest due to their applications for drug delivery, electronic displays and as micromotors. However, up till now, there is no single protocol capable of generating particles of "patchy" composition with a variety of well-defined and predictable shapes. To address this, in this submission we dispersed magnetic nanoparticles (MNPs) in a non-magnetic fluid containing monomer and crosslinker. This solution was added to the surface of Teflon, which was submerged in the solvent 2,2,4-trimethylpentane. Under these conditions a round, stable droplet was formed on the Teflon. Upon exposure to a permanent magnet, the MNPs self-assembled into clusters with a variety shapes and sizes. The shape and size of the clusters depended on the magnetic field strength, which we controlled by systematically varying the distance between the magnet and the droplet. Interestingly, the shape of the liquid droplet was also influenced by the magnetic field. Upon polymerization, the MNP patterns and the droplet shape was preserved. We also show that very complex MNP patterns and particle shapes could be generated by controlling the distance between the drop and both a magnet above and below the droplet. In this case, the resulting patterns depended on whether the magnets were attracting or repelling each other, which was capable of changing the field lines that the MNPs align with. Overall, this approach is capable of generating particles with predictable MNP patterns and particle shapes without the use of any templates or complex synthetic steps. Furthermore, by using a sprayer (or similar approaches, e.g., ink jet printing) this technique can be easily scaled up to produce many complex anisotropic particles in a short amount of time.
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