Synapt g2 si hdms instrument

The MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization—Time of Flight Mass Spectrometry) and MS-MS (tandem mass spectrometry) of YGB were 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 the MassLynx software, version 4.1 SCN916 (Waters Corporation, Wilmslow, UK). To act as a matrix, 2,5-dihydroxybenzoic acid solution (20 μg/μL) in 50% acetonitrile was used. The sample was dissolved in 50% methanol to a concentration of 20 μg/μL and then mixed with matrix solution in the proportion of 1:1 (v/v) [42 (link)]. Spectra were recorded in a positive ion mode. For MS/MS experiments, isolated precursor ions were fragmented using a collision voltage of 15 V. MS data were collected for 120 s. Mass spectra were assigned with multi-point external calibration using red phosphorous (Sigma Aldrich, St. Louis, MO, USA).

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)].

Prior to MS analysis, proteins were buffer exchanged into 200 mM ammonium acetate buffer pH 7.4 (Sigma) using Bio-Spin microcentrifuge columns (Bio-Rad Laboratories). Intact MS spectra were recorded on a Synapt G2-Si HDMS instrument (Waters Corporation) modified for high mass analysis and operated in ToF mode. Samples were introduced into the ion source using borosilicate emitters (Thermo Fisher Scientific). Optimized instrument parameters were as follows: capillary voltage 1.4 kV, sampling cone voltage 80 V, offset voltage 80 V, transfer collision voltage 25 V and argon flow rate 5 ml/min. Collision voltage in the trap was optimized between 50 and 110 V. Data was processed using MassLynx v.4.2 (Waters).

Reversed phase chromatography was performed using Waters AQUITY Ultra Performance LC system (Milford, MA, USA), with an analytical column BEH C18 1.7 µm, (2.1 × 100 mm, beds diameter: 1.7 µm). The column was heated to 40 °C and eluted with a gradient of solvents from 99% A and 1% B to 1% A and 99% B, where: A: water, with 0.1% formic acid; B: acetonitrile, with 0.1% formic acid.
The flow rate of the mobile phase was 0.4 mL/min. The sample volume was 5–10 µL. Samples were dissolved in 50% acetonitrile and were injected using AQUITY autosampler.
ESI-MS spectrometry was performed with SYNAPT G2-Si HDMS instrument (Waters Corporation, Milford, MA, USA) operating in positive ion mode. Acquisition of the data were performed at a range of 100–2000 m/z, using MassLynx software, version 4.1 SCN916 (Waters Corporation, Wilmslow, UK). Mass spectra were assigned with a multi-point external calibration using sodium iodide (Sigma-Aldrich, St. Louis, MO, USA). Mass spectrometer conditions were as follows: capillary voltage: 3.00 kV, sampling cone: 40 V, source offset: 80 V. Ion source temperature was established at 100 °C and desolvation temperature: 200 °C. Cone gas flow was set at 100 L/h and desolvation gas flow—800 L/h.

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.