Amazon speed

All reagents and solvents were purchased from Sigma Aldrich (Toluca, Mexico) and used without further purification. Reactions were monitored by thin-layer chromatography (TLC) on Merck F253 silica gel aluminum sheets, and spots were revealed with ultraviolet (UV) light (254 nm). NMR experiments were carried out in Varian NMR System (500 MHz and 125 MHz), Varian Mercury (300 MHz and 75 MHz) and Bruker ASCEND (600 MHz and 150 MHz). ¹H NMR and ¹³C spectra were assigned with the help of 2-D experiments (gHSQC and gHMBC). The chemical shifts (δ) are given in ppm. Mass spectra (MS) were recorded on a Bruker Amazon Speed (ESI). Infrared (IR) spectra were obtained on a Perkin Elmer FT-IR Spectrum 2000 spectrometer from the ENCB-IPN spectroscopy instrumentation center. Melting points were determined on an Electrothermal MEL-TEMP apparatus and are uncorrected. Microwave reactions were accomplished on a CEM Discovery SP apparatus.

Prior to MS analysis the extracts were dissolved in methanol. MS experiments were performed on a Dionex Ultimate 3000 UPLC system (ThermoFisher Scientific, Waltham, MA, USA) with PDA detector (stationary phase 100 mm ACQUITY UPLC BEH C18 1.7 µm column (Waters Corporation, Milford, MA, USA), mobile phase: linear gradient of [A] ddH2O + 0.1% formic acid / [B] acetonitrile + 0.1% formic acid, 5% to 95% at a flow rate of 0.6 mL/min). For mass detection the system was further coupled to either an amaZon speed (Bruker, Billerica, MA, USA) or LTQ Orbitrap XL mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) applying standard settings of positive ionization and a mass range detection of m/z 200 to 2000. The data were analyzed by the softwares Compass Data Analysis 4.1 (Bruker) or Xcalibur 3.0 (ThermoFisher Scientific).

Steroid compounds and culture supernatants were analysed by liquid chromatography coupled to mass spectrometry (LC-MS) using a Dionex Ultimate 3000 LC system with a UV/visible light diode array detector (ThermoFisher Scientific; Waltham, MA, U.S.A.), and an ion trap mass spectrometer (Amazon speed; Bruker, Bremen, Germany) with an electro-spray ion source (ESI) as described previously⁷⁰ . For the evaluation of measurements, MS- or UV-base peak chromatograms or extracted ion chromatograms with defined mass ranges were used as indicated. For analysing the degradation of CDC and the accumulation of intermediates in sand, samples were centrifuged at >16,000 × g for 5 min at room temperature, before the supernatants were used for LC-MS analyses. For analysing the degradation of CDC and the accumulation of intermediates in the presence of C. elegans, samples were centrifuged as described, stored at −20 °C, acidified (pH 1–2), cleaned from hydrophilic contaminants by organic extraction with ethyl acetate, and dissolved in methanol. Samples from soil experiments were also cleaned by organic extraction prior to the analysis by HPLC-MS. For that, 200 μl of samples were acidified with 30 μl of 1 M of HCl (pH 1–2) and extracted with 600 μl of ethyl acetate. Ethyl acetate was dried off, and the samples were dissolved in 150 μl of ethanol. All extracted samples were then analysed by LC-MS.

Chitosan oligomers were separated using a Dionex Ultimate 3000RS UHPLC system (Thermo Fisher Scientific, Waltham, USA) coupled to an evaporative light scattering detector (Model Sedex 90LT, Sedere, Alfortville Cedex, France) and an ESI-MSⁿ-detector (amaZon speed, Bruker, Bremen, Germany). Separation of the oligomers was achieved by hydrophilic interaction liquid chromatography (HILIC) using an Acquity UHPLC BEH Amide column (1.7 μm, 2.1 mm × 150 mm; Waters Corporation, Milford, USA) in combination with a VanGuard pre-column (1.7 μm, 2.1 mm × 5 mm; Waters Corporation, Milford, USA). The samples were split between ELSD and ESI-MSⁿ detectors using a 1:1 splitter (Accurate, Dionex Corporation, Sunnyvale, USA). All of the used UHPLC-ELSD-ESI-MSⁿ methods were based on the ones described by Cord-Landwehr et al. [17 (link)]. The injection volume was always 1 μl for undiluted samples and 2 μl for samples that were diluted with equal parts of 0.5 M NaOH to stop an enzymatic reaction. Data Analysis 4.1 software (Bruker, Bremen, Germany) was used for analysis of the results.

The extracts obtained from liquid cultures of Heimiomyces sp. were dissolved in acetone to yield a concentration of 10 mg/mL. Analysis of the samples was performed with an analytical HPLC device (Dionex UltiMate 3000 series, Sunnyvale, CA, USA) coupled to an ion trap mass spectrometer (amazon speed™ by Bruker). As mobile phase HPLC grade water and MeCN, both containing 0.1% of formic acid, were used. After injection of 2 μL of the samples, the separation was carried out over an ACQUITY-UPLC^® BEH C18 column (50 × 2.1 mm; particle size: 1.7 μm) (Waters) with a flow rate of 600 μL/min. The gradient started at 5% of MeCN, then increased to 100% MeCN in 20 min and remained for 5 min at 100%. To evaluate the obtained chromatograms, the appropriate analysis software (Data Analysis, version 4.4 by Bruker) was used.

Peptide digests were analyzed by electrospray ionization in the positive mode on an ion trap instrument Amazon Speed (Bruker, Bremen, Germany), using captive spray source. Two analytical replicates of every sample were done. Peptides were separated by nanoflow HPLC (NanoAdvance, Bruker, Bremen, Germany). UHPLC Nanotrap (100 μm i.d. × 25 mm long) packed with 200 A C₁₈ stationary phase (5 μm, C18AQ, Michrom) was used for peptide trapping. Analytical columns (100 μm × 150 mm long) packed with 200 A C₁₈ stationary phase (3 μm, C18AQ, Michrom) were coupled to the mass spectrometer (MS).
Peptide mixtures obtained after tryptic digestion were applied to the precolumn at a flow rate of 5 μL min⁻¹ in 2% (v/v) acetonitrile with 0.1% (v/v) formic acid. Peptides were eluted by a linear gradient of A (water, 0.1% formic acid) and B (acetonitrile, 0.1% formic acid), as follows: 0 min – A (98%), B (2%); 50 min – A (5%), B (95%); 50–55 min – A (5%), B (95%) with flow rate of 400 nL min⁻¹. Ion source conditions were optimized with a calibration solution according to the instrument provider. All MS survey scans were performed from m/z 400–1400 with enhanced resolution. Data analysis was performed by selection of the five most abundant precursors rejecting singly charged ions.