Gas-Phase Protonation Thermodynamics of Biological Lipids: Experiment, Theory, and Implications

Abstract

Phospholipids are important to cellular function and are a vital structural component of plasma and organelle membranes. These membranes isolate the cell from its environment, allow regulation of the internal concentrations of ions and small molecules, and host diverse types of membrane proteins. It remains extremely challenging to identify specific membrane protein–lipid interactions and their relative strengths. Native mass spectrometry, an intrinsically gas-phase method, has recently been demonstrated as a promising tool for identifying endogenous protein–lipid interactions. However, to what extent the identified interactions reflect solution- versus gas-phase binding strengths is not known. Here, the “Extended” Kinetic Method and ab initio computations at three different levels of theory are used to experimentally and theoretically determine intrinsic gas-phase basicities (GB, ΔG for deprotonation of the protonated base) and proton affinities (PA, ΔH for deprotonation of the protonated base) of six lipids representing common phospholipid types. Gas-phase acidities (ΔG and ΔH for deprotonation) of neutral phospholipids are also evaluated computationally and ranked experimentally. Intriguingly, it is found that two of these phospholipids, sphingomyelin and phosphatidylcholine, have the highest GB of any small, monomeric biomolecules measured to date and are more basic than arginine. Phosphatidylethanolamine and phosphatidylserine are found to be similar in GB to basic amino acids lysine and histidine, and phosphatidic acid and phosphatidylglycerol are the least basic of the six lipid types studied, though still more basic than alanine. Kinetic Method experiments and theory show that the gas-phase acidities of these phospholipids are high but less extreme than their GB values, with phosphatidylserine and phosphatidylglycerol being the most acidic. These results indicate that sphingomyelin and phosphatidylcholine lipids can act as charge-reducing agents when dissociated from native membrane protein–lipid complexes in the gas phase and provide a straightforward model to explain the results of several recent native mass spectrometry studies of protein–lipid complexes.

Detergent-free Lipodisq Nanoparticles Facilitate High-Resolution Mass Spectrometry of Folded Integral Membrane Proteins

“…In our tandem MS (Figure 3 C), a charge-reducing effect was observed for DMPC adducts but not for the highly anionic ether lipid aducts. 66 We propose that dissociation of DMPC molecules, with a net positive charge, is also responsible for the lower charge state of bR observed in the Lipodisq spectra compared with the OG micelles.…”

Electrospray ionization of native membrane proteins proceeds via a charge equilibration step

“…The picture is more complex for fos-choline: the structurally related lipid phosphatidylcholine has the highest gas-phase basicity of all common lipids and is prone to causing charge reduction upon dissociation from a protein complex, in line with our ndings for fos-choline. 40,41 However, the chargereducing effect of fos-choline detergents is concentrationdependent, even above the CMC and therefore this effect must not solely rely on interactions between protein and micelle. Interestingly, the amine oxide detergent LDAO as well as its head-group trimethylamine-N-oxide (TMAO) have been found to reduce the charge of protein ions through gas-phase collisions.…”

Mass Spectrometry Methods for Measuring Protein Stability

“…It is important to note that not all solubilization agents are equally effective in this endeavor, and screening detergents and solution conditions and optimizing instrument parameters are necessities for striking the delicate balance between the removal of solubilization agents and optimizing the stability of the native MP structure . However, with successful optimization, nMS has been used to study discrete lipid − and ligand binding (Figure A and F) events, as well as to quantify the thermodynamics associated with lipid binding − (Figure B) and specific protein–protein interactions associated with a wide range of MPs − and their functional assemblies ,,− (Figure C).…”