Apollo 2 electrospray source

DOM for non-targeted mass spectrometric analysis was extracted using solid-phase extraction cartridges following the procedure described in Dittmar et al.44 Samples were ionized by an Apollo II electrospray source (Bruker Daltonik GmbH, Bremen, Germany) in negative ion mode and measured on a SolariX (FT-ICR-MS; Bruker Daltonik GmbH) equipped with a 12 T refrigerated actively shielded superconducting magnet (Bruker Biospin, Wissembourg, France). Data evaluation started with internal calibration of the mass spectra with a suite of compounds repeatedly identified in marine DOM samples22 45 . The root mean square error of the internal calibration was below 0.100 ppm. Data evaluation was performed in the range m/z 200 to 600. For peaks with a signal to noise ratio higher than 3, molecular formulas were calculated in the mass accuracy range of ±0.5 ppm by considering the following elements: ¹H (0 to 90), ¹²C (0 to 60), ¹³C (0 to 1), ¹⁶O (0 to 35), ¹⁴N (0 to 4), ³²S (0 to 2), ³⁴S (0 to 1). The criteria summarized in Koch et al.46 (link) were applied for formula assignment. For the final dataset we focused on ions with a relative intensity (normalized to the highest peak in each spectra) of ≥3%, corresponding to a signal to noise ratio of ≥6.

A high-resolution mass spectrum of α-KBA was recorded with a 12 Tesla solariX FT-ICR-MS (Fourier transform ion cyclotron resonance mass spectrometer; Bruker Daltonics, Bremen, Germany) equipped with an APOLLO II electrospray source (Bruker Daltonics) and operated in the negative ionization mode. A diluted sample (β = 10 µg/mL in MeOH/water (7/3, v/v)) was infused into the MS with a constant flow rate of 120 µL/h. Spectra were acquired with a time-domain of 4 megawords and 30 scans were accumulated within an m/z range from 147 to 750. Calibration of the MS and ion source settings were the same as recently described [60 (link)]. The MS method achieved highest resolving power of 393,000 at m/z 469.332267 and superb mass accuracy in the ppb range.
All NMR (nuclear magnetic resonance) spectra of α-KBA were recorded on a 500 MHz Bruker Avance III HD spectrometer equipped with a CryoPlatform and a 5 mm TCI CryoProbe (Bruker Biospin, Karlsruhe, Germany). Spectrometer control and data processing were accomplished with Bruker TopSpin Ver. 3.6.1. The sample was measured in DMSO-d₆ and data were referenced to the remaining solvent signals at δ_H 2.50 and δ_C 39.51, respectively.

Ms4a15 OE and control were prepared as described [32 ]. For analysis, cells were resuspended in 800 µL methanol and transferred into beat tubes. Eppendorf cups were flushed additionally with 200 µL to transfer remaining cells. Cells were lysed using 2 × 15 s, below 4 °C (Precellys, Bertin) and centrifuged with 12,000 rpm for 15 min. The supernatant was immediately diluted 1:10 in methanol. Mass spectra were acquired on a 12 T solariX FT-ICR mass spectrometer (Bruker Daltonics) using an Apollo II electrospray source (Bruker Daltonics), in broad band detection mode with a time domain transient of 2 Megawords in positive and negative electrospray mode. The instrument was calibrated with a 1 ppm arginine solution. A mass error below 100 ppb was achieved. Injected velocity was set to 120 µL/h. Mass lists were generated with a signal-to-noise ratio (S/N) of four, exported, and combined to one data matrix by applying a 1 ppm window. Ions (m/z mass/charge) were annotated using MassTRIX allowing 1 ppm mass tolerance. Unidentified metabolites were annotated by elemental composition using mass-differences based network approach allowing 0.1 ppm mass tolerance [82 (link)].