Ltq orbitrap xl hybrid mass spectrometer

Chemicals and reagents were from Merck (Darmstadt, Germany). Evaporation of solvents was performed on a vacuum rotary evaporator (Rotavapor R-3000r, Buchi, Switzerland). FCPC was carried out on a Kromaton instrument equipped with a 1000-ml column, adjustable rotation of 200–2000 rpm and a preparative Laboratory Alliance pump with a pressure safety limit of 50 bar. NMR spectra in MeOD were recorded at 400 and 600 MHz (Bruker Advance III 600 MHz and DRX 400). 2D NMR experiments, including correlation spectroscopy (COSY), heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) were performed using standard Bruker microprograms. Electrospray ionisation mass spectrometry (ESI-MS) experiments were performed on a LTQ-Orbitrap XL hybrid mass spectrometer (Thermo-Scientific, Bremen, Germany). Analytical TLC was performed on Merck Kieselgel 60 F₂₅₄ or RP-8 F₂₅₄ plates. Spots were visualized by UV light (254 and 365 nm) or by spraying with sulfuric vanillin. The plates were then heated for 2 min at 110°C. Preparative TLC was conducted on PLC Silica gel 60 F254 plates (1 mm). The selected zones were scraped and extracted with ethyl acetate to separate the corresponding compounds. Column chromatography was performed on silica gel 70–230 mesh (63–200 μm). Size exclusion chromatography was performed on Sephadex LH-20.

Samples were re-solubilized in 5% [v/v] acetonitrile, 0.2% [v/v] formic acid in water and injected into an Ultimate 3000 nano-flow uHPLC-System (Dionex Co, Thermo Scientific, San Jose, CA) that was in-line coupled to the nanospray source of an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Scientific, San Jose, CA). Peptides were separated on an in-house packed emitter-tip column (75 µm ID PicoTip fused silica tubing (New Objectives, Woburn, MA) filled with C-18 material (5 µm bead size) to a length of 12 cm) and eluted by a 35 min. gradient starting at 5% [v/v] acetonitrile, 0.2% [v/v] formic acid and finishing at 80% [v/v] acetonitrile, 0.2% [v/v] formic acid in water at a flow rate of 500 nl/min.

In this study, U-HPLC-DAD/ESI-MS/MS analysis method was carried out to demonstrate and characterize the major constituents of PEESG. The analysis was performed on an Accela U-HPLC system (Thermo Fisher Scientific, San Jose, CA) coupled with LTQ Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA), fitted with an ESI source. Samples were separated on a reversed-phase Kinetex C18 column (100 mm × 2.10 mm, 1.7 μm, Phenomenex Inc., USA) using a flow rate of 0.3 mL/min at 25°C. The mobile phase consisted of eluent A (methanol) and eluent B (aqueous formic acid solution, 0.2%, v/v). A gradient program was used for elution: 0–30 min, A from 15% to 95%, B from 85% to 5%. Analytes were determined by ESI-MS/MS selected reaction monitoring in the negative ion mode. The triple quadrupole MS and spray chamber conditions were gas temperature, 300°C; drying gas, nitrogen at 10 L/min; nebulizer pressure, 15 psi; sheath gas temperature, 250°C; sheath gas flow, nitrogen at 7 L/min; capillary voltage, 4 kV.

All solvents were high performance liquid chromatography grade and were used without modification. All standard compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA). Formic acid, methanol and acetonitrile were obtained from Merck (Merck KGaA, Darmstadt, Germany). Distilled water was produced by a Milli-Q reagent-grade water system (Millipore; Billerica, MA, USA). UPLC was performed on a Thermo Fisher Accela system and mass spectrometry was performed on an LTQ Orbitrap XL hybrid mass spectrometer (both from Thermo Fisher Scientific, Franklin, MA, United States).

Cultures for exoproteome analysis were prepared by spinning the cells (3000 g at room temperature during 15 min) and gently re‐suspending them in fresh media at a final concentration of 3 × 10⁷ cell ml⁻¹. Cultures were then left to grow in standard conditions to cell densities of 10⁸ cells ml⁻¹. At this point, cultures were subjected to centrifugation at 3000 g for 15 min at room temperature. Supernatants were carefully removed and then gently filtered through 0.22 μm pore size filter units (Sterivex‐GV, Millipore) to eliminate any remaining cells. Proteins in the remaining milieu were concentrated and purified by precipitation with trichloroacetic acid and run on SDS‐PAGE as previously described (Christie‐Oleza and Armengaud, 2010). Trypsin in‐gel proteolysis of the entire exoproteome was performed for the shotgun proteomics analysis as recommended (Hartmann et al., 2014). NanoLC‐MS/MS experiments were performed using a LTQ‐Orbitrap XL hybrid mass spectrometer (ThermoFisher) coupled to an UltiMate 3000 LC system (Dionex‐LC Packings). Conditions used were those previously described (de Groot et al., 2009).

Culture supernatants of control cells and those exposed to fumed silica NPs during 24 h were collected and pooled (v = 2.5 mL per condition, biological triplicates). Proteins from each sample were precipitated with 10% trichloroacetic acid after the addition of a one-quarter volume of trichloroacetic acid at 50% (w/vol). The solutions were vortexed and then incubated on ice for 10 min. The samples were centrifuged at 16,000 g for 10 min. at 4°C. The resulting pellet was dissolved in 45 μl 1X LDS (Invitrogen). These samples were heated at 99°C for 5 min. and then loaded onto a 10% NuPAGE gel (Invitrogen) for a short electrophoresis in MOPS buffer. The polyacrylamide bands containing the whole exoproteomes were processed as previously described [47 (link)]. The resulting peptides (10 μl of the 40 μl generated with the procedure) were analyzed with an LTQ-Orbitrap XL hybrid mass spectrometer (ThermoFisher) coupled to an UltiMate 3000 Nano LC System (Dionex-LC Packings), as described previously [48 (link)].