Maldi plate

The procedure for in-gel digestion of protein spots from Coomassie Blue stained gels was carried out as described in [9 (link)]. In brief, protein spots were excised from stained gels and cut into pieces. The gel pieces were washed for 1 h at room temperature in 25 mM ammonium bicarbonate buffer, pH 7.8, containing 50% (v/v) acetonitrile (ACN) and dehydrated in a SpeedVac for 10 min and rehydrated in 10 μL (20 ng/μL) of sequencing grade trypsin solution (Promega, WI). After incubation in 25 mM ammonium bicarbonate buffer, pH 7.8, at 37°C overnight, the tryptic peptides were extracted with 5 μL of 0.5% TFA containing 50% (v/v) ACN for 40 min with mild sonication. The extracted solution was reduced to 1 μL in a vacuum centrifuge. The resulting peptide solution was desalted using a reversed-phase column [10 (link)] and subjected to mass spectrometric analysis. A GEloader tip (Eppendorf, Hamburg, Germany) constricted was packed with Poros 20 R2 resin (PerSpective Biosystems, MA). After an equilibration with 10 μL of 5% (v/v) formic acid, the peptide solution was loaded on the column and washed with 10 μL of 5% (v/v) formic acid. The bound peptides were eluted with 1 μL of α-cyano-4-hydroxycinnamic acid (CHCA) (5 mg/mL in 50% (v/v) ACN/5% (v/v) formic acid) and dropped onto a MALDI plate (96 × 2; Applied Biosystems, CA).