Esquire hct ion trap mass spectrometer

The protein contents in the LDL(+) and LDL(−) were evaluated by LC-ESI MS/MS on an Esquire HCT ion trap mass spectrometer (Bruker, Bremen, Germany) coupled with a nanoHPLC system (Ultimate, LcPackings, Netherlands) in the Laboratory of Proteomics at the Vall de Hebron Institute of Research (VHIR, Barcelona, Spain), as described in [28 (link)]. The lipoproteins were delipidated and solubilized according to Karlsson et al. [48 (link)], with minor modifications. The proteins were then digested with trypsin, and the proteomic analysis was performed by LC-ESI MS/MS using 5 µg of protein [28 (link)]. Proteins were identified using Mascot (Matrix Science, London, UK) to search the UniProtSwissProt 57.0 human database.

The purified fraction of interest was solubilized in HPLC grade MeOH at a 1 mg/mL concentration. It was analyzed using an Agilent 1100 HPLC (Santa Clara, CA, USA) mounted with an Inertsil ODS-3 3 µm 3 × 100 mm column and using the following gradient: acetonitrile: mQ water (85:15, solvent B) and mQ water (solvent A) + 0.2% formic acid and 12 mM ammonium formate. One minute of 53% B was followed by a linear increase of B to 100% at 17 min and an isocratic elution at 18 min. The gradient was then reversed at 6 min followed by a stabilization after 3 min at a flow rate of 0.5 mL/min. The injected volume was 10 µL.
Then using an ESI-ion trap mass spectrometer (Esquire HCT ion trap mass spectrometer; Bruker, Rheinstetten, Germany), the fraction of interest was characterized. The ESI was operated in the positive mode. Mass spectrometer parameters were set as follows: capillary voltage -4500 V; endplate offset −500 V; nebulizer pressure 50 psi; dry gas flow rate 10 L/min; dry gas temperature 300 °C; skimmer 20.6 V; capillary exit 300 V; oct 1 DC 10 V; oct 2 DC 2.79 V; Lens 1–5.2 V; lens 2–72 V; oct RF 300 Vpp; trap drive 92.8. The scan range was optimized at 300–1250 m/z.

Thin-layer chromatography was performed using Merck TLC Silica gel 60 F254 or TLC Silica gel 60 RP-18 F254S. UV light (254 nm and 354 nm) and/or a 10 % H₂SO₄ stain were used to visualize the spots on TLCs. Column chromatography was performed on silica gel provided by Brunschwig (32–63 mesh, 60Å) prepacked columns. NMR measurements were carried out on a Bruker Avance III HD 500 MHz spectrometer (^1H: 500 MHz, ¹³C: 125 MHz). Deuterated solvents were obtained from Cambridge Isotope Laboratories. HRESI-MS was performed on a MicrOTOF-Q mass spectrometer (Bruker, Germany). ESI-MS reaction monitoring was carried out using a Bruker esquire HCT Ion trap mass spectrometer. IR spectra were recorded on a Bruker FT-IR Tensor II using a Golden Gate diamond ATR system. Optical rotations were measured on a Perkin Elmer Polarimeter 241 using the sodium lamp (589 nm) and a 10 cm long cuvette. Microwave heating was performed on a Biotage Initiator Microwave using Biotage microwave vials. UV/VIS spectra were recorded on a UV/VIS Lambda 25,190–1100 nm. Irradiations were performed using Rayonet photochemical reactors.

Culture supernatants obtained after centrifugation of the culture were filtered on 0.2 µm MiniUniPrep filter (Whatman). When following “method A”, the filtered supernatants were analyzed on an Atlantis T3 column (250 mm × 4.6 mm, 5 μm, 30 °C) using an Agilent 1200 HPLC instrument equipped with a quaternary pump. Samples were eluted with isocratic 0.1% HCOOH in H₂O (solvent A) / 0.1% HCOOH in CH₃CN (solvent B) (95:5) at 1 ml/min for 5 min followed by a gradient to 100% solvent B over 45 min. Molecules that were present in the supernatant were detected by an Alltech 3300 evaporative light scattering detector (ELSD) (Grace).
When necessary, an analysis of the cell content was made. The mycelium pellet obtained after centrifugation of 2 ml of culture, was washed twice with 2 ml of water, suspended in 400 µl of water and broken down with glass beads using the FastPrep-24 apparatus (MP Biomedicals). A cell-free extract was obtained after centrifugation (13,000 g, 4 °C, 30 min) to remove cell debris.
LC-MS analyses were performed using an Agilent 1100 HPLC coupled via split system to an Esquire HCT ion trap mass spectrometer (Bruker) set in positive and negative modes. Multiple analyses were made. Some of them were made using “method A”, some other using the conditions described by^{15 (link)} for CDPS 1–47 and called “method B”.