Red phosphorous

1,5‐Diaminonaphthalene, red phosphorous, and ammonium formate were purchased from Sigma‐Aldrich (St Louis, MO). Tissue‐Tek^® OCT compound 4583 was sourced from Sakura Finetek (Torrance, CA). Unless otherwise stated, all other reagents were purchased from Sigma‐Aldrich.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS) was performed using a Waters SYNAPT G2-Si HDMS instrument (Waters Corporation, Milford, MA, United States) equipped with a 1 kHz Nd:YAG laser. Phospholipids isolated from the wild-type cells and the mutant cells were dissolved in chloroform/methanol (2/1, v/v) at a concentration of 10 mg mL^–1. One microliter of the sample was mixed with one microliter of the matrix, transferred into the target plate, and allowed to dry. The matrix solution was prepared from 2,5-dihydroxybenzoic acid (DHB, Sigma-Aldrich) (10 mg mL^–1, chloroform/methanol 2/1, v/v). Spectra were recorded in positive and negative ion modes. Data acquisition was performed using MassLynx software version 4.1 SCN916 (Waters Corporation, Wilmslow, United Kingdom). For the MS/MS experiments, the isolated precursor ions were fragmented using collision voltage of 35 V for PC, PE, PG, and 60 V for CL. Data were collected for 120 s for each ion separately. Mass spectra were assigned with a multi-point external calibration using red phosphorous (Sigma-Aldrich). The regiochemistry of phospholipids was determined based on the intensity ratio of sn-2/sn-1 carboxylate anions. This ratio was higher than 1 for PC, PG, and PE (Fang and Barcelona, 1998 (link)).

MALDI-TOF-MS spectrometry was performed with a Waters Synapt G2-Si HDMS instrument (Waters Corporation, Milford, MA, USA) equipped with a 1 KHz Nd:YAG laser system (355 nm wavelength). Acquisition of the data was performed using MassLynx software, version 4.1 SCN916 (Waters Corporation, Wilmslow, UK). Mass spectra were assigned with multi-point external calibration using red phosphorous (Sigma-Aldrich, Saint Louis, MO, USA) in the range of 100–3000 Da. The sample was dissolved in water containing 0.1% TFA at a concentration of 20 µg/µL, subjected to ultrasonication for 5 min, and desalted on a piece of Parafilm^® with a few grains of Dowex 50WX8–100 (Serva, Heidelberg, Germany) converted into the H⁺ form. The desalted sample was mixed with a DHB matrix (2,5-dihydroxybenzoic acid (Sigma-Aldrich, Saint Louis, MO, USA), dissolved in 50% methanol, 20 µg/µL), and 1 µL of the mixture was transferred into the target plate wells. Spectra in the positive ion mode were recorded.

The LPS and oligosaccharide samples were analyzed with matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) using a Waters SYNAPT G2-Si HDMS instrument (Waters Corporation, Milford, MA, USA) equipped with a 1 kHz Nd:YAG laser system. Acquisition of the data was performed using MassLynx software version 4.1 SCN916 (Waters Corporation, Wilmslow, UK). Mass spectra were assigned with a multi-point external calibration using red phosphorous (Sigma) and recorded in the negative ion mode. Phenol-soluble LPS and oligosaccharide samples (both at a concentration of 15 µg/µL) were suspended in a water/methanol (1:1, v/v) solution (containing 2 mM EDTA for the LPS sample) and dissolved by ultrasonication. After desalting with the use of cation exchange beads (Dowex 50WX8-200; Sigma), one microliter of each sample was transferred onto a well plate covered with a thin matrix film and allowed to dry at room temperature. The matrix solution was prepared from 2′,4′,6′-trihydroxyacetophenone (THAP) (200 mg/mL in methanol) mixed with nitrocellulose (15 mg/mL) suspended in 2-propanol/acetone (1:1, v/v) in a proportion of 4:1 (v/v), according to the published method [65 (link),66 (link)].

Polyacrylonitrile (PAN, average Mw ~ 150,000) was purchased from Sigma-Aldrich (USA). N, N dimethylformamide (DMF; 99.5%) was purchased from Tokyo Chemical Industry Co. Inc. (Tokyo, Japan). Red Phosphorous (RP, purity ≥ 97%) was purchased from Sigma-Aldrich (Italy). Plastic syringes and needles (21G 1/2) were purchased from Terumo (Tokyo, Japan). LiPF₆ (1 mol L⁻¹) in ethylene carbonate (EC)/dimethyl carbonate (DMC) (1:1 v/v%) solution was purchased from Sigma-Aldrich (Italy). Cr2032 coin cells components were purchased from MTI Corporation, KJ Group (USA).

MALDI-TOF mass spectrometry was performed with a SYNAPT G2-Si HDMS instrument (Waters Corporation, Milford, MA, USA) equipped with a 1 KHz Nd:YAG laser system (355 nm wavelength). Acquisition of the data was performed using MassLynx software version 4.1 SCN916 (Waters Corporation, Wilmslow, UK). Mass spectra were assigned with a multi-point external calibration using red phosphorous (Sigma). The lipid A samples were dissolved in chloroform/methanol (3:1, v/v) at a concentration of 10 µg/µL, and 2 mM of EDTA was added. A sample (1 µL) was transferred into the target plate wells covered with a thin matrix film. The matrix solution was prepared from 2’,4’,6’-trihydroxyacetophenone (THAP) (200 mg/ml in methanol) and mixed with nitrocellulose (NC) (15 mg/ml, suspended in 2-propanol/acetone (1:1, v/v)) in a proportion of 4:1 (v/v), as previously described by Silipo and co-workers [33 (link)]. Spectra were recorded in positive and negative ion modes.

For MALDI–TOF mass spectrometry analysis, a 0.5 M 2,5-dihydroxybenzoic acid (DHB) solution in methanol containing 0.1% trifluoroacetic acid (TFA) was used as matrix. The extract of MLB total lipids was diluted in chloroform/methanol (2:1, v/v) to a concentration of 5 μg/μL and directly applied onto the target plate as 1 μL droplets, followed by the addition of 1 μL DHB matrix solution. After drying and crystallization at room temperature, samples were analyzed by MALDI–TOF–MS and MS–MS.
All MALDI–TOF mass spectra were acquired on a Waters SYNAPT G2-Si HDMS instrument (Waters Corporation, Milford, MA, USA) equipped with a 1 kHz Nd:YAG laser system. Acquisition of the data was performed using MassLynx software version 4.1 SCN916 (Waters Corporation, Wilmslow, United Kingdom). Spectra were recorded in the positive ion polarities. For MS² experiments, isolated precursor ions were fragmented using a collision voltage of 50–60 V. Data were collected for 120 s for each ion separately. Mass spectra were assigned with multipoint external calibration using red phosphorous (Sigma-Aldrich, St. Louis, MO, USA).