Native MS Analysis of Membrane Proteins

Membrane protein samples were buffer-exchanged into aqueous ammonium acetate (200 mM, pH 7.4 adjusted with ammonium hydroxide) supplemented with 0.5% C₈E4 (AA C₈E₄) for AmtB or 0.065% C₁₀E₅ (AA C₁₀E₅) for the other proteins using a centrifugal desalting column (Micro Bio-Spin 6, Bio-Rad). The protein–lipid mixtures were loaded into a gold-coated glass emitter (prepared in-house) and introduced into a Q Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo). The instrument parameters were set as follows: For AmtB, capillary voltage of 1.50 kV; capillary temperature of 200 °C; Collision-Induced Dissociation (CID) of 50 V; Collision Energy (CE) of 80 V; trapping gas pressure setting to 3.0; source DC offset of 20 V; injection flatapole DC of 10 V; inter flatapole lens of 6 V; Bent flatapole DC of 4 V; transfer multipole DC of 6 V. For TRAAK and TREK2, capillary voltage of 1.50 kV; capillary temperature set to 300 °C; CID of 50 V; CE of 50 V; trapping gas pressure set to 5.0; source DC offset of 40 V; injection flatapole DC of 8 V; inter flatapole lens of 4 V; bent flatapole DC of 3 V; transfer multipole DC of 3 V. The MS data collected from native MS were deconvoluted using UniDec and Protein Metric software.^{43,44 (link)} Mass error was determined for the bound lipids based on the center of each peak.^{45 (link)}