1260 infinity liquid chromatograph

Protein samples (~12 μM) for LC-MS were incubated with either an 8-fold or 40-fold molar excess of formaldehyde to EcFrmR tetramer concentration at room temperature. After 3 min, reactions were quenched with 10 mM glycine. Samples were loaded onto an Agilent 1260 Infinity liquid chromatograph fitted with an Agilent Extended C18 column (2.1 mm × 50 mm) and eluted with a gradient of 5-95% acetonitrile in 0.1% formic acid at 400 μl min⁻¹ over 8 min. The eluate was directly coupled to an Agilent 6530 Q-ToF mass spectrometer fitted with an electrospray ionisation (ESI) source for determination of the masses of species detected in the chromatograph.
For ICP-MS, EcFrmR (200 μM) in 50 mM Tris (pH 8.0) buffer containing 0.5 M NaCl or buffer was incubated with concentrated nitric acid (1:1 ratio) at 60 °C for 1 h. Samples were cooled, diluted with dH₂O (up to 10 ml) and filtered before analysis on a Perkin Elmer Nexlon ICP-MS system. Ions were quantified using a dilution series of certified multi-element reference standard (Sigma-Aldrich). Counts per second values for elements in the buffer and protein samples were then compared with the calibration curve to determine actual concentrations.

The purity of the starting materials and the synthesized compounds, as well as the analysis of reaction mixtures, was monitored by TLC on Silica gel 60 F₂₅₄ plates (Merck, Albany, GA, USA) using chloroform, methanol, or their mixtures as eluent. Chromatograms were developed using UV irradiation, treatment with iodine vapor, or heating the plate. The purity of the obtained compounds is confirmed by HPLC analysis. HPLC analysis was performed using an Agilent 1260 Infinity liquid chromatograph equipped with a UV detector in combination with an Agilent 6230 TOF LC/MS detector. The melting points of the resulting compounds were determined on a Stuart SMP30 instrument. ¹H NMR spectra were recorded on spectrometers “Bruker AV400”, “Bruker DRX-500”, and “Bruker AV600” operating frequency 400.16, 500.13, and 600.13 MHz, respectively. ¹³C NMR spectra were recorded on a Bruker AV400 spectrometer with an operating frequency of 100.62 MHz. The spectra were recorded at 20 °C using DMSO-d6 as a solvent. For synthetic purposes, commercially available solvents and reagents (SigmaAldrich (St. Louis, MO, USA), Merck, Acros Organics (Geel, Belgium)) were used.

The extract solution was filtered through a 0.45 μm filter membrane before injection. The gel chromatograph analysis was executed on an Agilent 1260 infinity liquid chromatograph incorporated with a laser light–scattering detector at 254 nm (Li et al., 2014 (link)). The Milli–Q water was used as the mobile phase, and eluted at a flow rate of 1 mL min^–1. The overlapping peaks were analyzed by peak–fitting technique. Due to the limitation of pores, the evolution of large molecule substances was faster than small molecule substances. Higher–molecular weight (HMW), MMW and LMW fractions were classified according to retention time less than 6 min, 6–8 min and more than 8 min, respectively, following the procedure described by He et al. (2019) (link).