1 palmitoyl 2 oleoyl sn glycero 3 phospho 1 rac glycerol popg

Prior to MS analysis, both native and citrullinated peptides were diluted ten times in 1 M ammonium acetate, pH 7.5, or 0.1% acetic acid, pH 4.5. For lipid binding experiments, the peptides were diluted in 1 M ammonium acetate, pH 7.5, containing 4 mM of detergent lauryldimethylamine N-oxide (LDAO) to a final concentration of 5 µM each. In brief, zwitterionic (neutral charge) 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3[phospho-rac-(1-glycerol)] (POPG) were obtained from Avanti Polar Lipids (Alabaster, Alabama, USA) with a concentration of 800 µM each were prepared in Milli-Q water. For binding studies, each phospholipid was added to the mixture of peptides in LDAO to a final concentration of 80 µM. Mass spectra were recorded on an Orbitrap Fusion (Thermo Fisher Scientific, Waltham, MA) equipped with an offline nano-electrospray source. Samples were introduced using gold-coated Proxeon borosilicate capillaries (Thermo Scientific, Waltham, MA). Spectra were recorded in a positive ionization mode with a capillary voltage of 1.8 kV and a source temperature of 80 °C. Data were analyzed using the Xcalibur software 3.0 package (Thermo Scientific). Student’s T-test for paired samples with equal variance was performed in Microsoft Excel.

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), and CHOL (ovine
wool) were purchased from Avanti Polar Lipids (Alabaster, AL). An
extract of natural porcine lung surfactant, poractant alfa (Curosurf,
Chiesi Farmaceutici, Parma, Italy), enriched with CHOL (10 wt %) was
used in control experiments. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
labeled with Atto633 (DOPE–Atto633) was obtained from ATTO-TEC
(Siegen, Germany). Organic solvents of spectroscopic grade used for
the preparation of lipid working solutions were supplied by Merck
(Darmstadt, Germany). Phosphate buffered saline (PBS) (Sigma-Aldrich,
St. Louis, MO) prepared with Mili-Q water (Millipore, USA), with addition
of 0.2 mM ethylenedinitrilotetraacetic acid (EDTA) (Sigma-Aldrich,
St. Louis, MO), was used as a subphase in the experiments. All chemicals
were used without further purification.

1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho(1′-rac-glycerol)
(POPG), purchased from Avanti Polar Lipids and stored in a chloroform
solution at −20 °C, was used to prepare synthetic lipid
liposomes. A nitrogen stream was used to evaporate the chloroform,
and the sample was further desiccated for 1 h in a vacuum. The lipids
were then hydrated in PBS supplemented with 2 mM MgCl₂ to
give a final lipid concentration of 4 mg/mL. Single unilamellar vesicles
were made by lipid extrusion through a 50 nm pore polycarbonate membrane,
and samples were stored for up to 2 weeks at 4 °C.

Large unilamellar vesicles (LUVs) were prepared using methods previously described (14 (link)). Briefly, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) (Avanti Polar Lipids) were combined in a 3:1 ratio, respectively, in chloroform and dried to a film before water washing and lyophilizing into a powder. This powder was resuspended in Tris-Cl buffer (10 mM Tris, 10 mM NaCl, pH 7), freeze-thawed 5× (dry ice – 50 °C water bath) and extruded through a mini-extruder fitted with a 0.2 μm Whatman Nuclepore polycarbonate membrane filter (Cytiva) at least 15× to form LUVs. Where applicable, 1-palmitoyl-2-(6,7-dibromo)stearoyl-sn-glycero-3-phosphocholine (PC-Br (6 (link), 7 (link))) or 1-palmitoyl-2-(11,12-dibromo)stearoyl-sn-glycero-3-phosphocholine (PC-Br (11 (link), 12 (link))) (Avanti Polar Lipids) were substituted for POPE at the given percentages and incorporated into LUVs as described.