Hct ultra

SUN2-containing protein complexes were purified, resolved by 10–20% SDS-PAGE, and stained with a mass spectrometry-compatible Silver Stain MS kit (Wako Pure Chemical Industries Ltd.). Corresponding bands were cut out for trypsinization. Tandem mass spectra of the trypsinized peptides were obtained using a nano LC (UltiMate 3000; Thermo Scientific Dionex, CA, USA) equipped with an L-column 2 C18 (150 mm × 0.075 mm i.d. analytical column, containing 3-µm particles; Chemicals Evaluation and Research Institute, Japan), coupled with a nano-ESI (electrospray ionization)-IT (ion trap)-MS (mass spectrometer) equipped with a nano-ESI ion source (HCTultra; Bruker Daltonik GmbH, Germany). The raw data files were analyzed using Mascot MS/MS Ion Search (Matrix Science, London, UK) and searched against the Swiss-Prot database of human proteins.

Amygdalin and cyanidin 3-O-glucoside were obtained from Sigma-Aldrich and indol-3-yl-methyl glucosinolate from Cfm Oskar Tropitzch GmbH. Dhurrin, prunasin, epiheterodendrin and epidermin were chemically synthesized46 (link). LC-MS analysis was performed using a Zorbax SB-C18 column on an Agilent 1100 Series LC coupled to a Bruker HCT-Ultra ion trap mass spectrometer as described previously8 (link). Compounds were localised in extracted ion chromatograms as sodium adduct ions: dhurrin (m/z 334), prunasin (m/z 318), amygdalin (m/z 480), epidermin (m/z 284), epiheterodendrin (m/z 284), cyanidin 3-O-glucoside (m/z 449), indol-3-yl-methyl glucosinolate (m/z 493). Relative quantification was based on peak area using Bruker-DataAnalysis 4.0 (Bruker Daltonik).

All manipulations were performed under N₂ using standard Schlenk techniques and dried, deoxygenated solvents. Acetonitrile was refluxed over calcium hydride. THF and diethylether were dried over sodium benzophenone. The ligands p-tert-butylcalix[6]areneH₆ and p-tert-butylcalix[8]areneH₈ were obtained from TCI UK and were dried in vacuo at 80 °C for 6 h prior to use. CoBr₂ was purchased from Sigma Aldrich and was dried for 12 h at 100 °C in vacuo (affording the green form) prior to use. IR spectra (nujol mulls, KBr windows) were recorded on a Nicolet Avatar 360 FT IR spectrometer. Elemental analyses were performed by the elemental analysis service at the Department of Chemistry, the University of Hull. Mass spectra were recorded as LCMS, on a Bruker Daltonics HCT Ultra which uses Hystar 3.2. Data was processed in Data Analysis 4.2. For each sample, a 1 mg mL⁻¹ stock solution was prepared, which was serially diluted to 10 μg mL⁻¹ in dry acetonitrile. Example spectra are shown in Fig. S1 (of 6) and S2 (of 7) in the ESI.†

Peptides separations were performed on an Ultimate3000 HPLC (Thermo). Tryptic peptides were loaded onto a trap column (PepMap100 C18, 5 μm, 100 Å, 300 μm ID, 5 mm) using buffer A (flow: 20 μl/min) and separated using reverse-phase C18 analytical column (PepMap100 C18, 3 μm, 100 Å, 75 μm i.d., 15 cm) with a linear gradient of buffer B (95% acetonitrile, 4.9% H2O, 0.1% formic acid) ranging from 0 to 50% within 70 min (flow: 300 nl/min). Eluted peptides were analysed by nanoESI ion trap mass spectrometer (HCTultra, Bruker) set to isolate and fragment the top 5 most abundant precursors. Electrospray voltage was set to 2000V and analysis mass ranges were 250–1500 m/z for MS and 200–3000 m/z for MS/MS. DataAnalysis software version 3.4 from Bruker was used to generate mgf files from raw data, with a precursor intensity threshold of 3.10⁵. The mgf files were submitted to local Mascot server using both PlasmoDB v13 (14 jan 2015, 5542 entries) and human SwissProt (6 march 2013, 20329 entries) databases. Search parameters were the following: MS and MS/MS mass tolerance = 0.5 Da; missed cleavages = 1; fixed modification = Carbamidomethyl (C), variable modification = Oxidation (M): FDR < 1% (search against the reversed merged database).

Product samples were analyzed by direct infusion on a Bruker HCT Ultra ion trap. Ionization was performed by electrospray with methanol as ionizing phase in the negative reflector mode.

The Liquid chromatography equipment (Agilent 1100 series) comprises of a binary pump, an auto sampler having a 100 µL capacity loop and a Diode Array Detector with a range from 200 to 600 nm. The detector recorded at 254, 280 and 320 nm, which is the best absorption wavelength for polyphenolic compounds. Chromatographic separation was performed using the same gradient method used in the LC-TOF analyses. A 5 μm diphenyl column of 250 × 3 mm i.d. (Varian, Darmstadt, Germany) with 500 µL/min flow rate of solvent was used. The LC equipment was connected with Ion-trap mass spectrometer, which was fitted with an ESI source (Bruker Daltonics HCT Ultra, Bremen, Germany) operating in full scan auto MSⁿ mode to obtain fragment ions. Tandem mass spectra were acquired in Auto-MSⁿ mode (smart fragmentation) using a ramping of the collision energy. Maximum fragmentation amplitude was fixed to 1 V. MS operating conditions (negative mode) had been optimized with a capillary temperature of 365 °C, a dry gas flow rate was of 10 L/min, and a nebulizer pressure of 10 psi. 3 µL of the filtered extract was injected into the system. The software used in this system was Agilent Chemstation.