Concentrator plus vacuum centrifuge

2-D gel electrophoresis spots of interest were excised and transferred into 1.5 ml sterile LoBind tubes (Eppendorf) for in-gel protein digestion. Gel pieces were de-stained with 100 μl of 50% (vol/vol) ACN/50 mM ammonium bicarbonate (NH4HCO3) for 10 min at room temperature on tube-rotating mixers. De-staining was repeated for at least three times or until the gel pieces became colourless. Gel pieces were dehydrated in 100% acetonitrile and dried in a Concentrator plus vacuum centrifuge (Eppendorf). The dried gel pieces were rehydrated with 50 μl of a 1:40 dilution of trypsin (15 ng/μl, sequencing grade) in 50 mM NH4HCO3 and incubated at 4°C for 1 h. After digestion, excess trypsin was removed and 20 μl of 50 mM NH4HCO3 was added to cover the gel pieces. The tubes were then incubated at 37°C for 16 h. Final protein extraction was performed on ice by sonication as follows: 2 s (sonication) and 4 s (rest) for 20 min in total. The supernatant was then dried using a Concentrator plus vacuum centrifuge (Eppendorf) at 45°C for 20 min. MALDI TOF (matrix-assisted laser desorption ionization–time of flight) mass spectrometry was performed using a TOF/TOF 5800 system (AB SCIEX) for peptide mass fingerprinting and protein identification.

Cells were separated from the supernatant via centrifugation of 1 mL in Eppendorf cups at 13,000 rpm for 10 min. A total of 500 µL of the supernatant was transferred to separate glass Pyrex^® tubes for preparation of the metabolite extracts. An equivalent volume of 500 µL ethyl acetate was added to each tube and extracted using a multirotator (Grant-bio PTR-60, Grant Instruments Ltd., Shepreth, UK) for 2 min at 50 rpm with a change in direction every 3 s for better mixing of the 2-phase system. This was repeated once with removal of the organic phase and addition of fresh solvent in between each round. The ethyl acetate extract was collected in LC glass vials and evaporated to dryness in a Concentrator Plus vacuum centrifuge by Eppendorf. The vacuum–high vapor (V-HV)-mode was used at 30 °C for 40 min. A total of 75 µL of MS-grade methanol was added to each dry extract to dissolve the extracted compounds.