Deciphering Solution and Gas-Phase Interactions between Peptides and Lipids by Native Mass Spectrometry

Many biological processes depend on the interactions between proteins and lipids. Accordingly, the analysis of protein–lipid complexes has become increasingly important. Native mass spectrometry is often used to identify and characterize specific protein–lipid interactions. However, it requires the transfer of the analytes into the gas phase, where electrostatic interactions are enhanced and hydrophobic interactions do not exist. Accordingly, the question remains whether interactions that are observed in the gas phase accurately reflect interactions that are formed in solution. Here, we systematically explore noncovalent interactions between the antimicrobial peptide LL-37 and glycerophospholipids containing different headgroups or varying in fatty acyl chain length. We observe differences in peak intensities for different peptide–lipid complexes, as well as their relative binding strength in the gas phase. Accordingly, we found that ion intensities and gas-phase stability correlate well for complexes formed by electrostatic interactions. Probing hydrophobic interactions by varying the length of fatty acyl chains, we detected differences in ion intensities based on hydrophobic interactions formed in solution. The relative binding strength of these peptide–lipid complexes revealed only minor differences originating from van der Waals interactions and different binding modes of lipid headgroups in solution. In summary, our results demonstrate that hydrophobic interactions are reflected by ion intensities, while electrostatic interactions, including van der Waals interactions, determine the gas-phase stability of complexes.


Dynamic light scattering
The mean hydrodynamic diameter of detergent-lipid micelles was determined using a Litesizer 500 particle size analyzer (Anton Paar, Graz, Austria).For this, 100 µl of detergent-lipid micelles were prepared as described and analyzed in a 3 x 3 mm ultra-micro cuvette (Hellma Analytics, Müllheim, Germany).The particles were irradiated with a semiconductor laser diode at 658 nm.The following instrument settings were applied: measuring angle, side scatter (90°); temperature, 25 °C; measurement time, automatic; filter, automatic; focus, automatic; material, phospholipids; solvent, 154 mM NaCl.The mean hydrodynamic diameter was determined from size distribution histograms using the software Kalliope (Anton Paar, Graz, Austria).

Circular dichroism spectroscopy
Circular dichroism (CD) spectroscopy was performed using a J-810 spectropolarimeter (JASCO, Groß-Umstadt, Germany).For this, 50 µl of a 1 mg/ml solution of LL-37 in 1 x PBS as well as in 20 mM AmAc in the presence and absence of 0.5 % (w/v) C8E4 were analyzed in a 0.1 mm quartz cuvette at 20 °C.
The following instrument parameters were applied: wavelength, 190-240 nm; scanning mode, continued; scan number, 64 scans; scan speed, 50 nm/min; response, 1 s; and data pitch, 1 nm.The raw data was reduced to data points at HT voltage below 600 V as the signal to noise ratio is lower at high dynode voltages.CD spectra were smoothed using a binomial filter and the reference spectrum of the buffer was subtracted using the Spectra Manager software (JASCO).The ellipticity was converted to mean residue ellipticity (Δε) as described previously 1 .Masses of LL-37-lipid complexes are given in Table S1.S1.

S9
3. Supporting Tables Table S1: Masses of LL-37 and LL-37-lipid complexes determined by native MS.The figure number, the peptide and lipid, the theoretical mass and the experimental mass of assigned peaks are given.
Theoretical masses were calculated from the molecular weight of associated lipids and the amino acid sequence of LL-37.Experimental masses and the corresponding error were determined using MassLynx v4.1.

Figure S1 :
Figure S1: Structure of LL-37.(A) Solution structure of LL-37 in the presence of SDS micelles (pdb ID 2K6O).Hydrophobic (orange), basic (blue), acidic (red) and uncharged polar (green) amino acids are indicated.(B) Helical wheel projection of LL-37 with Heliquest 2 .The hydrophobic and the hydrophilic interfaces are indicated (red line).(C) CD spectra of LL-37 in 20 mM AmAc in the presence (black) and in the absence (purple) of 0.5 % (w/v) C8E4 as well as in 1 x PBS (pink).

Figure S4 :
Figure S4: Dissociation of LL-37-PE and LL-37-PC complexes.Native MS of LL-37 in the presence of C8E4 with PE 14:0/14:0 (left) or PC 14:0/14:0 (right) at different collisional voltages.Charge states and peaks corresponding to the LL-37 monomers are assigned.The intensity of LL-37-lipid complexes decreases with increasing collisional voltage.At higher collisional voltages (i.e., above 70 V) an increase in background signal was observed.