Apex qe

To analyze the pnWTA bound to small PGN fragments, electrospray ionization fourier-transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) was performed on a 7 Tesla APEX Qe instrument (Bruker Daltonics, Bremen, Germany) using negative-ion mode and a water/propan-2-ol/7 M triethylamine/acetic acid mixture (50:50:0.06:0.02 v/v/v/v) as solvent, as described previously^{6 (link)}. MS analysis of hydrazine-treated LTA was done on a Q Exactive Plus (Thermo Scientific, Bremen, Germany) in negative-ion mode using the same solvent. A Triversa Nanomate (Advion, Ithaca, USA) ion source was used with a spray voltage set to −1.1 kV. The mass scale was calibrated externally with glycolipids of known structure, and all spectra were charge deconvoluted. The given mass numbers refer to the monoisotopic mass of the neutral molecules.

A Bruker Apex-Qe Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Bruker Daltonics, Bremen, Germany) was employed for most of the APFD experiments. The instrument was equipped with a 9.4 T superconducting magnet and an ESI-to-MALDI switchable Dual Source. The mass spectrometer was controlled by the Bruker ApexControl software (V 3.0.0) and data analysis was performed using the Bruker DataAnalysis software (V 4.3).
External mass calibrations were established in ESI mode by either using Agilent Tune Mix (G1969-85000) for the m/z 200–1800 range^{44 ,45} or arginine [Arg_n–H]^– cluster ions for the m/z 150–1200 range.^{52 ,53 (link)} Mass accuracy was generally in the order of 3 ppm.

The crude products (QAS-phenylboronic acid derivatives) were analyzed using a Thermo Separation HPLC system with a UV detection (210 nm) on a Vydac Protein RP C18 column (4.6 × 250 mm, 5 μm), with a gradient elution of 0%–80% B in A (A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile/H₂O, 4:1) for 40 min (flow rate 1 mL/min). The main reaction product was purified by a preparative reversed-phase HPLC on a Tosoh (Tosoh Tokyo, Japan) TSKgel with an ODS-120 T column (21.5 mm × 300 mm), using a linear gradient 10%–20% of B for 40 min (PhB-K(QAS)-NH₂) and 2%–12% of B for 40 min (PhB(QAS)-GGG-NH₂), flow rate 7.0 mL/min, UV detection at 220 nm. The fractions were collected and lyophilized. The identities of the products were confirmed by MS analysis using Bruker Apex-Qe (Bremen, Germany) Ultra 7 T FT-ICR mass spectrometer equipped with an electrospray ionization source.

The analysis of the m/z ratio molecule contained in the extracted photoluminescent fraction was characterized by a Bruker brand Mass Spectrometer (MS) model Apex-Qe.

An equivalent amount of bacterial cultures grown up to an OD₅₉₅ of 0.5 were harvested by centrifugation. Pellets were resuspended in 1X sample buffer, boiled for 10 min, followed by digestion with Proteinase K to obtain whole cell lysates. Equivalent portions of such whole cell lysates were applied to a 14% SDS-Tricine gel. After the electrophoresis LPS was transferred by Western blotting. Immunoblots were probed for LPS amounts using the WN1-222-5 monoclonal antibody [50 (link)] and revealed by chemiluminescence kit from Thermo Scientific (Warsaw, Poland).
Bacterial cultures (400 mL to 1 L) were grown in phosphate-limiting medium, harvested by centrifugation and pellets were lyophilized. For the LPS analysis, lyophilized material was dispersed in water by sonication and resuspended at a concentration of 2 mg/mL and LPS was extracted by the phenol/chloroform/petroleum ether procedure [68 (link)]. Electrospray ionization-Fourier transform ion cyclotron (ESI-FT-ICR)-mass spectrometry was performed on intact LPS in the negative ion mode using an APEX QE (Bruker Daltonics, Breman, Germany) equipped with a 7-tesla actively shielded magnet and dual ESI-MALDI. LPS samples were dissolved at a concentration of ∼10 ng/μL and analyzed as described previously [11 (link),69 (link)]. Mass spectra were charge deconvoluted, and mass numbers given refer to the monoisotopic peaks.

Electrospray ionization Fourier-transform ion cyclotron (ESI FT–ICR) mass spectrometry was performed on either intact LPS or using glycerophospholipids and lipid A mixture in the negative ion mode. LPS samples were dissolved at a concentration of ∼10 ng/μL and analyzed, as previously described [33 (link)]. For the acquisition of mass spectra, an APEX QE (Bruker Daltonics, Bremen, Germany) equipped with a 7-tesla actively shielded magnet and dual ESI-MALDI was used. Mass spectra were charge-deconvoluted, and the mass numbers given refer to the monoisotopic peaks. Mass calibration was done externally using well-characterized compounds of known structures, as described previously [19 (link),81 (link)].