Influence of Alkylammonium Acetate Buffers on Protein–Ligand Noncovalent Interactions Using Native Mass Spectrometry

Abstract

We investigate the influence of three volatile alkylammonium acetate buffers on binding affinities for protein-ligand interactions determined by native electrospray ionization-mass spectrometry (ESI-MS). Four different types of proteins were chosen for this study. A charge-reduction effect was observed for all the cases studied, in comparison to the ions formed in ammonium acetate solution. When increasing the collision energy, the complexes of trypsin and the ligand were found to be more stable when sprayed from alkylammonium acetate buffers than from ammonium acetate. The determined dissociation constant (K) also exhibited a drop (up to 40%) when ammonium acetate was replaced by alkylammonium acetate buffers for the case of lysozyme and the ligand. The prospective uses of these ammonium acetate analogs in native ESI-MS are discussed in this paper as well. Graphical Abstract ᅟ.

Nanoscale Ion Emitters in Native Mass Spectrometry for Measuring Ligand–Protein Binding Affinities

“…The +7 and +8 charge states (Figure S5) for unbound and ligand-bound lysozyme were the most abundant ions, which is consistent with the charge-state distributions reported by native MS previously. 6,7,38 By reducing the tip diameter from 2000 to 250 nm, the K d value decreased from 9.4 ± 0.3 to 7.6 ± 0.1 μM (Table S2). The K d values obtained using the 850, 500, and 250 nm ESI emitters (7.6–7.8 μM) are in excellent agreement with values reported in the literature based on measurements using ultraviolet spectroscopy (6.6 μM), 36 fluorescence spectroscopy (8.6 μM), 51 ESI-MS (9.9 μM), 7 and isothermal titration calorimetry (11.1 μM).…”

“…The interactions between proteins and ligands are crucial to proper cellular function. , The structures, functions, and interactions of protein–ligand complexes can be significantly affected by salts. − Specific metal ion cofactors can regulate the bioactivity of proteins . In native mass spectrometry (MS), ligand–protein interactions are normally stabilized using volatile salts at high ionic strengths to rapidly and directly measure the mass, binding stoichiometry, and relative ligand–protein binding affinities with high sensitivity. − However, most biochemical approaches to probe protein–ligand interactions, including nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, and optical spectroscopy, use nonvolatile salts that can more accurately reflect the in vivo environment of the protein–ligand complex. However, nonvolatile salts and common biological buffers readily adduct to proteins ions to result in broad spectral peaks that have deleterious effects on mass spectra by lowering the sensitivity and signal-to-noise ratios and increasing background chemical noise .…”

A Guide to Native Mass Spectrometry to determine complex interactomes of molecular machines

“…Native MS experiments are usually performed using volatile buffers such as ammonium acetate [ 64 , 65 ], ethylenediammonium diacetate or alkylammonium acetate buffers [ 66 ]. Unlike other types of ESI‐MS analysis, neither acidic conditions nor organic solvents are used.…”

Label‐free Bio‐affinity Mass Spectrometry for Screening and Locating Bioactive Molecules

“…The binding affinity of ligands was dependent on the pI of the protein: when pI > pH, a decrease in Kd was found with increasing AmAc concentration, whereas if not, the inverse was the case. In addition, Zhuang, Gavriilidou, & Zenobi (2017) expanded the work of Gavriilidou et al by showing three volatile alkylammonium acetate buffers exhibited pronounced stabilization and a charge‐reducing effect on lyz‐NAG3, trypsin–pefabloc and carbonic anhydrase II–chlorothiazide. In these cases, alkylammonium acetate buffers are better choices than AmAc in native MS for studying protein–ligand affinity.…”