Cells were collected at day 2 of culture, from 10 µm MeJA treated and ethanol control suspensions, ground in liquid nitrogen and precipitated with the same volume of 50% trichloroactetic acid (w/v) on ice for 1 h. After centrifugation for 45 min at 4°C at 23 700g (Beckman J2-HS centrifuge, rotor JA 20) the pellets were washed with 3 volumes of 100% acetone three times as above. After washing in 2 volumes of ethyl ether and centrifugation for 10 min at 4°C at 1000g (Beckman J2-HS centrifuge, rotor JA 20) the powder was air dried for 30 min and 30 mg of powder was resuspended in 1.3 ml of liquid phenol (pH 7.9) added with 1% (v/v) 2-mercaptoethanol. Liquid extracts were recovered by centrifugation at 20 000g at 4°C for 30 min, extracted twice with 50 mm Tris-HCl pH 8, 1% (w/v) SDS, and then precipitated with 5 volumes of acetone on ice for 2 h. Samples were centrifuged 45 min at 6°C at 20 000g, the pellet washed twice with 1.3 ml of acetone and once with 1 ml of diethyl ether, air dried for 10 min and resuspended in 400 µl of rehydration buffer containing 2% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 7 m urea, 2 m thiourea, 1% (v/v) IPG buffer pH 3–10 (Amersham Bioscience, Chalfont St. Giles, UK). After centrifugation samples were loaded onto 18 cm Immobiline DryStrip pH 3–10 NL (nonlinear) (Amersham Bioscience, Chalfont St. Giles, UK), overnight at 18°C, then focused using a 18 cm pHaser with chiller block (Genomic Solutions, Huntingdon, UK) at a total of 85 KVh over a period of 23–24 h at 20°C. Strips were then equilibrated in equilibration buffer (0.063 m Tris-acetate, 3.3% (w/v) SDS, 6 m Urea, 30% (v/v) glycerol, traces bromophenol blue) added with 0.8% DTT (w/v) for 30 min and then in equilibration buffer added with 2.5% (w/v) iodoacetamide for 30 min. Second dimension was run on 10% Duracryl gels in a Tris-tricine buffer (top running buffer: 0.2 m Tris base, 0.2 m tricine, 0.4% (w/v) SDS; bottom running buffer: 25 mm Tris-acetate), in an Investigator 2-D large format 5-gel electrophoresis tank (Genomic Solutions, Huntingdon, UK) at 20 W per gel, at 20°C, for 4–5 h, until the bromophenol blue dye front was within 2 cm from the bottom of the gel. 2-D gels were fixed in 40% (v/v) methanol, 10% (v/v) acetic acid 1 h, stained with Sypro Ruby fluorescent dye (Bio-Rad Laboratories, Hercules, CA, USA) for 12 h, and washed in 10% (v/v) methanol, 6% (v/v) acetic acid for 1 h. Gel images were recorded using a ProExpress multiwavelength fluoroimager (Perkin Elmer Lifesciences, Cambridge, UK) and analysed using the ProteomeWeaver 2.1 software from Definiens AG (Munich, Germany). Spots were picked with an ‘Investigator’ ProPic spot picker and trypsin digested with an ‘Investigator’ ProGest in gel digestion unit (both from Genomic Solutions, Huntingdon, UK) as described by Speicher et al. (2000).
Samples were submitted to the Institute of Food Research and John Innes Center Joint Proteomics Facility (Norwich, UK). The acidified digests were spotted directly onto a thin layer of matrix on a stainless steel target made by mixing three parts of a saturated solution of α-cyano-4-hydroxycinnamic acid (CCA) in acetone with one part of a 1 : 1 mixture of acetone : isopropanol containing 10 mg ml−1 nitrocellulose. Digests were externally calibrated to yield data of better than 50 ppm mass accuracy. Analysis of peptide digests was carried out on a Reflex III MALDI-ToF (Bruker Ltd, Coventry, UK) with Scout 384 ion source using a nitrogen laser (λ = 337 nm) to desorb/ionise the matrix/analyte material from the sample substrate. Ions generated in this way were allowed to drift for the short delayed extraction time setting, before being accelerated by a potential of +25 kV. Spectra were acquired in reflectron mode. Peptide fingerprints were searched against the National Center for Biotechnology Information-non-redundant database, taxonomy Viridiplantae, using the searching algorithm Mascot (http://www.matrixscience.com).
Peptides generated from tryptic digestion were loaded at high flow rate onto a reverse-phase trapping column (0.3 mm i.d. × 1 mm, containing 5 µm C18 100 Å PepMap packing, LC Packings, the Netherlands) and eluted through a reverse-phase capillary column (75 µm i.d. × 150 mm column, containing Symmetry C18 300 Å packing, Waters Ltd, Elstree, UK) directly into the nano-electrospray ion source of a quadrupole time-of-flight mass spectrometer (Q-ToF2, Micromass UK Ltd, Manchester, UK).
Fragment ion spectra were searched using the MASCOT search tool (Matrix Science Ltd, London, UK) against a weekly updated copy of the SPTrEMBL (UNIPROT) database using appropriate parameters. Peptides that were not identified by database search were sequenced de novo using the PepSeq software (Micromass, Manchester, UK).