Multiprobe 2

Concentrated >30 kDa fractions of bacterial supernatants were re-suspended in 0.1% ProteaseMax (Promega), 50 mM ammonium bicarbonate and 10% acetonitrile. Protein concentrations were determined using the BCA kit (Pierce). 5 μg of each sample were incubated for 30 min at 50 °C followed by 10 min bath sonication at room temperature. Samples were centrifuged and the supernatant was directly subjected to a tryptic digestion protocol carried out by a liquid handling robot (MultiProbe II, Perkin Elmer).

Each gel lane was divided into 20 bands, which were de-stained in 50 mM ammonium bicarbonate and 50% acetonitrile. Tryptic digestion was performed by a liquid-handling robot (MultiProbe II, Perkin Elmer), including protein reduction in 10 mM DTT and alkylation in 55 mM iodacetamide. Gel pieces were dehydrated in 100% acetonitrile, trypsin was added to a final concentration of 13 ng/μl, and the pieces were digested over night at 37 °C. Extracted peptides from consecutive bands were pooled according to their protein levels, resulting in two pools for each lane.

After incubation time, fermentation broths from the liquid media were extracted by adding equal volume of acetone [10 (link),11 (link),12 ] or equivalent volume of methyl-ethyl-ketone (MEK) for solid fermentation, as previously described [30 ,41 (link)], using a Multiprobe II robotic liquid handler (PerkinElmer, Waltham, MA, USA) and shaking at 220 rpm for 1 h. Acetone, miscible with water, has been described as the solvent of choice for whole broth extractions of liquid submerged fermentations [10 (link),11 (link),12 ], whereas MEK, immiscible with water, extracts just the region of medium to low polarity compounds, but has been reported to be more successful on ensuring maximum extraction of SM for solid state fermentations [9 (link)]. The samples were centrifugated, and 12 mL of supernatant from each vial was transferred to glass tubes containing 0.6 mL of DMSO and mixed. The amount of 5 mL of water was added to the solid fermentation extracts and organic solvents, and parts of water were evaporated under a heated nitrogen stream to a final volume of 3 mL (80/20 water/DMSO solution), along with a final concentration of 2 × WBE [10 (link)]. Each fermentation batch included extracts from control media to discriminate their components.

Spots of interest were excised from 2D gels and washed several times with water and dried for a few minutes. Trypsin digestion was performed overnight with a dedicated automated system (MultiPROBE II, Perkin-Elmer). The gel fragments were subsequently incubated twice for 15 min in a 0.1% CH₃CN solution in water to allow extraction of peptides from the gel pieces. Peptide extracts were then dried and dissolved in starting buffer for chromatographic elution, consisting of 3% CH₃CN and 0.1% HCOOH in water. Peptides were enriched and separated using lab–on–a–chip technology (Agilent, Massy, France) and fragmented using an on–line XCT mass spectrometer (Agilent). The ESI LC–MS/MS data were converted into DTA– format files that were further searched for proteins with MASCOT Daemon (Matrix Science [40] ). For protein identification, two strategies were employed to mine the maximum data. Measured peptides were searched in the NCBI nr–protein sequence database, viridiplantae (green plants [41] ), and in the Brassica EST database (Brassica Genome Gateway 2007, [42] ). Proteins with two or more unique peptides matching the protein sequence with a score >53, as defined by MASCOT, were considered as a positive identification. The spectra of each peptide were verified manually. In cases where protein identification data were lacking, BLAST analysis was performed [43] .

Caudal fin clips and blood samples, both approximately 2 mm³, were digested in proteinase K (Bio Basic Inc., Markham, Ontario, Canada) at 55°C for 8 h. A glassmilk protocol modified from [45 (link)] was used to extract the DNA using a Perkin Elmer Multiprobe II plus liquid handling system (Perkin Elmer, Waltham, MA, USA). DNA quality and quantity were checked against a standard using gel electrophoresis on 2% agarose gel.

10μg of extracted protein from each sample were dissolved in 0.1% ProteaseMax, 50 mM ammonium bicarbonate and 10% acetonitrile. Resulting protein solutions were incubated for 30 minutes at 50°C followed by an additional bath soncication of 10 min at room temperature. Samples were centrifuged and directly subjected to a tryptic digestion protocol carried out by a liquid handling robot (MultiProbe II, Perkin Elmer). This included protein reduction in 5 mM DTT at 56°C and alkylation in 15 mM iodacetamide for 30 min at room temperature in the dark. Trypsin was added in an enzyme to protein ratio of 1:30 and digestion was carried out over night at 37°C.

After protein spots excision from 2D gels, the excised fragments were washed several times and dried in a SpeedVac centrifuge for few minutes. Trypsin digestion (with 10 µL of a 10 ng/µL trypsin solution) was performed overnight with a dedicated automate (MultiPROBE II, Perkin Elmer). Thereafter, gel fragments were subsequently incubated twice for 15 min in a H₂O/CH₃CN (1∶1) solution to extract the peptides from the gel. The peptide extracts were then dried and solubilised in starting buffer for chromatographic elution, consisting of CH₃CN 3%/HCOOH 0.1% in water.