Mass Spectrometry Reveals the Direct Action of a Chemical Chaperone

Abstract

Despite their fundamental biological importance and therapeutic potential, the interactions between chemical chaperones and proteins remain difficult to capture due to their transient and nonspecific nature. Using a simple mass spectrometric assay, we are able to follow the interactions between proteins and the chemical chaperone trimethylamine- N-oxide (TMAO). In this manner, we directly observe that the counteraction of TMAO and the denaturant urea is driven by the exclusion of TMAO from the protein surface, whereas the surfactant lauryl dimethylamine- N-oxide cannot be displaced. Our results clearly demonstrate a direct chaperoning mechanism for TMAO, corroborating extensive computational studies, and pave the way for the use of nondenaturing mass spectrometry and related techniques to study chemical chaperones in molecular detail.

Generation of Charge-Reduced Ions of Membrane Protein Complexes for Native Ion Mobility Mass Spectrometry Studies

“…Membrane proteins are ionized using nano electrospray ionization (nESI) where droplets are produced that undergo cooling as well as evaporative processes that increase local salt/buffer ion concentrations, exclude water, and crowd molecules in the droplets [25,26]. Here, we report the unique properties of TMAO to charge reduce membrane proteins ionized by nESI, which is consistent with a recent report showing significant charge reduction of soluble proteins [27]. Beyond charge reduction, IM measurements provide evidence of protein structuring in the presence of TMAO.…”

“…Importantly, high concentrations of TMAO did not significantly compromise mass spectral resolution indicating TMAO easily releases from the protein during desolvation ( Figure S2) unlike other charge-reducing small molecules, such as E 4 , that result in peak broadening owing to non-specific adduction to the protein [19]. Titration of TMAO reveals a concentration dependent charge reduction ( Figure 1C) similar to that shown for soluble proteins [27]. A compaction of the charge state distribution was also observed from six down to three charge states at the highest concentration of TMAO.…”

“…Increased activation energy regimes were able to be used without significant perturbation to protein arrival time distributions. TMAO has recently been shown to counteract urea denaturation on a soluble protein [27], implying the osmolyte could be useful for both soluble and membrane protein complexes. IM-MS has revealed that ions generated using TMAO are compact and display narrow arrival time distributions.…”

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors

“…In addition, we reasoned that changing the basicity of the linker between the OGD head and tail, e.g. ether/carbamate < triazole, would allow us to effectively tune charge states of membrane proteins in mass spectra-a feature that is of enormous interest within the emerging native MS community [20][21][22][23][24] . Having synthesized these detergents we now evaluate their ability to extract membrane proteins, retain subunit interactions, and lipid binding properties, as well as to produce favorable characteristics for native mass spectra.…”

A new azobenzene-based design strategy for detergents in membrane protein research

“…2 Protein charge reduction has been identied as a key parameter in this process, because it lowers the disruptive impact of Coulomb interactions on protein structure. [3][4][5] Protein charge reduction can be achieved in many ways, for example, by using basic solution additives, [3][4][5] detergents, 4,6,7 or by treating the electrospray plume with acetonitrile vapour. 3 The extent of charge reduction that is needed for retaining native structural features of a protein complex is difficult to generalize and needs to be determined experimentally.…”