Axima cfr mass spectrometer

Grains of Yukinkomai, Yukinosei and Todorokiwase (100 mg) at 4 DAF were extracted with 8 m urea, 1% (w/v) CHAPS detergent, 10 mm ethylene diamine tetraacetic acid (EDTA) and 5 mm phenylmethylsulfonyl fluoride and centrifuged at 10 000 g for 10 min at 4 °C. The supernatants were precipitated with 10% (w/v) trichloroacetic acid and resolved with 9 m urea, 3% (w/v) IGEPAL detergent, and 2% (v/v) 2‐mercaptoethanol and then used for gel‐based proteomics. The procedures of 2D polyacrylamide gel electrophoresis (2D‐PAGE) and matrix‐assisted laser desorption ionization time‐of‐flight MS (MALDI‐TOF‐MS) were essentially identical to the previous reports (Kaneko et al., 2011; Nanjo et al., 2004). In 2D‐PAGE, the 1st dimension used isoelectric focusing with ampholine (pH 3.5–10) and the 2nd dimension used sodium dodecyl sulphate (SDS)‐PAGE with 16% separating gel. The 2D gels were stained with Coomassie brilliant blue R‐250 (Nanjo et al., 2004). The protein spots excised from the gels were digested by trypsin using standard procedures (Awang et al., 2010). MALDI‐TOF‐MS was carried out with a matrix of α‐cyano‐4‐hyrdoxycinnamic acid in an AXIMA‐CFR mass spectrometer (Shimadzu Corp.) and an Autoflex III TOF/TOF mass spectrometer (Bruker BioSpin, Yokohama, Japan; Kaneko et al., 2011).

Reagents were purchased from commercial sources (Aldrich) and used without further purification. Triethylamine (TEA) and THF were distilled and purged with Ar before use. TCNE and TCNQ were purchased from Aldrich, and all other reagents were purchased from commercial sources and used as received.
¹H NMR spectra of the samples were recorded with a Varian 400 MHz instrument (Palo Alto, CA, USA) in CDCl₃ using the residual solvent resonance of CHCl₃ at 7.26 ppm relative to SiMe₄ as internal reference. FT‐IR spectra were recorded in KBr pellets using a PerkinElmer LR‐64912C spectrophotometer (Waltham, MA, USA). Matrix‐assisted laser desorption ionization time‐of‐flight mass spectra (MALDI‐TOF‐MS) were recorded on a Shimadzu AXIMA‐CFR mass spectrometer (Kyoto, Japan). All UV/Visible spectra were recorded on a HITACHI U‐3010 spectrophotometer (Tokyo, Japan). SEM images were obtained on a Jeol JSM‐5400/LV (Tokyo, Japan) with accelerating voltage of 15 kV.

The mass spectra were recorded in linear positive ion mode over the 2,000–12,000 m/z range using an AXIMA‐CFR mass spectrometer from Shimadzu Biotech (Kratos, U.K.) equipped with a nitrogen laser (337 nm). The time pulse length of the laser was 3 ns. The maximum laser fluency was 60 mJ per pulse. The full laser power was indicated on the instrument as 180 arbitrary units (a.u.; 6 mW). The irradiated spot size was approximately 150 μm in diameter. An external calibration was performed using standard mixtures of peptides and proteins (TofMix, PepMix, LaserBio, France) and standard bacterial extracts (BTS standard) from Bruker BioSpin AG (Fällanden, Switzerland).