DOI: 10.5281/zenodo.7778567
title: Lipid-polymer nanoparticles to probe the native-like environment of intra-membrane rhomboid protease GlpG and its activity

description: Polymers can facilitate detergent-free extraction of membrane proteins into nanodiscs (e.g., SMALPs, DIBMALPs), incorporating both integral membrane proteins as well as co-extracted native membrane lipids. Lipid-only SMALPs and DIBMALPs have been shown to possess a unique property; the ability to exchange lipids through ‘collisional lipid mixing’. Here we expand upon this mixing to include protein-containing DIBMALPs, using the rhomboid protease GlpG. Through lipidomic analysis before and after incubation with DMPC or POPC DIBMALPs, we show that lipids are rapidly exchanged between protein and lipid-only DIBMALPs, and can be used to identify bound or associated lipids through ‘washing-in’ exogenous lipids. Additionally, through the requirement of rhomboid proteases to cleave intra-membrane substrates, we show that this mixing can be performed for two protein-containing DIBMALP populations, assessing the native function of intramembrane proteolysis and demonstrating that this mixing has no deleterious effects on protein stability or structure
description: Mass spectrometric analysis was performed essentially as described previously26,27. Lipid extraction from nanodiscs was performed according to Bligh and Dyer, with modifications. Briefly, an estimated 5 nmol total lipid in 240 µL water and internal standards (1.25 µl EquiSPLASH and 6.25 µl cardiolipin (CL) mix I, Avanti Polar Lipids, corresponding to a standard lipid mix molar amount of: 18:1(d7) Chol Ester (76 pmol), 15:0-18:1-d7-PC (66.4 pmol), 15:0-18:1-d7-PE (70.3 pmol), 15:0-18:1-d7-PI (59 pmol), 15:0-18:1-d7-PS (64.4 pmol), 15:0-18:1-d7-PG (65.44 pmol), C15 Ceramide-d7 (94.2 pmol), d18:1-18:1(d9) SM (67.8 pmol), 15:0-18:1(d7) DAG (85 pmol), 15:0-18:1(d7)-15:0 TAG (61.6 pmol), CL 57:4 (40.41 pmol), CL 61:1 (36.9 pmol), CL 80:4 (33.86 pmol), CL 86:4 (30.82 pmol)) was mixed with 0.9 mL of chloroform/methanol (1:2 (v/v)) for 10 min. After addition of 0.3 mL chloroform and 0.3 mL H2O, the sample was mixed again for 10 min, and phase separation was accelerated by centrifugation (1000 g, 3 min). The lower chloroform phase was carefully transferred to a clean glass vial. The upper water phase was mixed with 10 µL 1 M HCl and 300 µL chloroform for 10 min. After phase separation, the lower chloroform phase was carefully transferred to the glass vial with the chloroform phase from the first extraction. The solvent was evaporated by a gentle stream of argon at 37°C. Lipids were dissolved in 10 mM ammonium acetate in methanol, transferred to Twin.tec PCR plate sealed with Thermowell sealing tape and analyzed on a QTRAP 6500 triple quadrupole mass spectrometer (SCIEX) equipped with nano-infusion splay device (TriVersa NanoMate with ESI-Chip type A, Advion). The quadrupoles Q1 and Q3 were operated at unit resolution. PC analysis was carried out in positive ion mode by scanning for precursors of m/z 184.1 at a collision energy (CE) of 37 eV. PE and PG measurements were performed in positive ion mode by scanning for neutral losses of 141 Da and 189 Da at CEs of 25 eV and 30 eV, respectively. CL species were identified in positive ion mode by scanning for precursors of the masses (m/z 465.4, 467.4, 491.4, 493.4, 495.4, 505.5, 519.5, 521.5, 523.5, 535.5, 547.5, 549.5, 551.5, 573.5, 575.5, 577.5, 579.5, 601.5, 603.5, 605.5, 607.5, 631.5, 715.5 and 771.5 Da) corresponding DAG-H2O fragments as singly charged ions at CEs of 40-50 eV. Mass spectra were processed by the LipidView Software Version 1.2 (SCIEX) for identification and quantification of lipids. Lipid amounts (pmol) were corrected for response differences between internal standards and endogenous lipids. Correction of isotopic overlap in CL species was performed as described previously23. MS/MS scans to determine acyl-chain compositions in PE and PG were performed in negative ion mode by scanning for precursors of the masses of acyl chains (m/z 253.2, 255.2, 267.2, 269.2, 281.2, 283.2, 295.2) at a CE of -45 eV. The raw data and mass-spectra for samples discussed in this work can be found at the doi:10.5281/zenodo.7778567.