Esi source

The HPLC determination of DMAT was performed using the Waters 2695 Separations module equipped with a thermostable autosampler (5 °C), a column module (35 °C), and a Waters 2996 diode array detector (DAD). The separation of the compounds was achieved using a VDSphere PUR C18-M-SE (4.6 × 150 mm, 5 µm) (Agilent technologies, Palo Alto, CA, USA) column. For HPLC-MS measurements, the HPLC instrument was coupled with a MicroTOF-Q-type Qq-TOF MS instrument equipped with an ESI source from Bruker (Bruker Daltoniks, Bremen, Germany). The flow rate and run time were 1.0 mL/min and 12 min, respectively. The active component DMAT was detected with DAD at 260 nm and MS. For the separation of the compounds, isocratic eluent composed of 25% methanol, 40% AcN/water (9/1) containing 0.1% trifluoroacetic acid (TFA), and 35% water was used.

The reconstituted peptides were used for shotgun proteomics experiments for the identification of endogenous peptides. The peptides were separated using micro-LC (Thermo UltiMate 3000 HPLC System; Dionex, Sunnyvale, CA, USA) through an analytical column (Supelco Sigma-Aldrich, St. Louis, MO, USA; Ascentis Express C18, 25 cm×4.6 mm, 2.7 μm) coupled with ESI source (Bruker Daltonics, Germany) spray in Maxis-HD qTOF (Bruker, Germany) mass spectrometer. The elution was performed with a flow rate of 150 μL/min and a continuous gradient of 5% to 75% ACN over 135 min. In the solvent system, solvent A was 100% water with 0.1% formic acid, and solvent B was 100% ACN with 0.1% formic acid. Data were acquired in the data-dependent mode in a mass spectrometer that operated automatically switching between MS and MS/MS acquisition. The precursor ion MS spectra scan range of 200 to 2,200 (m/z) was used in the Q-TOF with resolution R = 75,000. The six most abundant precursor ions were searched for detection of different masses during acquisition and selected for fragmentation using collision-induced dissociation with a fixed cycle time of 3 s along with 2 min of release for exclusion filter (Q-TOF processing software; Bruker Daltonics, Germany).

MS of hLCN2-R-NTD utilized a 7 T Fourier transform ion cyclotron resonance instrument equipped with an ESI source (Bruker). Proteins were desalted as described previously (26 (link)) and electrosprayed from 1 μm solutions in 1:1 H₂O/CH₃OH at pH 2.5 (adjusted by the addition of CH₃COOH). From ESI MS spectra with internal calibration using polyethylene glycol with an average molecular mass of ∼1000 (PEG 1000), the measured mass (most abundant isotopic peak) of hLCN2-R-NTD after refolding and removal of the N-terminal His₆ tag and the TEV cleavage site by TEV protease was 11,050.20 Da, which agrees to within 0.8 ppm with the calculated mass of the 106-residue protein with two disulfide bonds (most abundant isotopic peak, 11050.21 Da). A quantitative analysis of the measured, and calculated isotopic profiles revealed that two disulfide bonds were formed in >99% of the protein (2S ensemble), and one disulfide bond was formed in the remaining fraction (<1%, 1S ensemble).