6340 ion trap mass spectrometer

Protein spots of interest were manually excised from colloidal Coomassie blue-stained 2D gels and digested. Briefly, gel fragments were washed three times for 20 min in destain solution containing 50% (v/v) methanol and 50 mmol ammonium bicarbonate/L. The excised spots were then air-dried for 1 h, which was followed by in-gel digestion in 30 µL of a digestion buffer containing 50 mmol/L ammonium bicarbonate and 6 ng/µL sequencing grade bovine trypsin (Roche Diagnostics, Meylan, France). After overnight digestion, the aliquots were treated with 50% (v/v) acetonitrile and 5% (v/v) formic acid. Speed-vas-dried peptide extracts were resuspended in 10 µL of 5% (v/v) acetonitrile and 0.1% (v/v) formic acid and then analyzed with a nanoflow liquid chromatograph LC1200 system coupled to a Q-TOF 6520 or to a 6340 Ion Trap mass spectrometer equipped with a nanospray source and a HPLC-chip cube interface (Agilent Technologies, Les Ulis, France). Briefly, peptides were enriched and desalted on a 40 nL C18 reversed-phase trap column and separated on a Zorbax C18 column (75 µm inner diameter x 43 mm long, 5 µm particle size and 30 nm pore size; Agilent Technologies). Peptides are eluted at 400 nL/min using a 17 min (for Q-TOF) or 9 min (for Ion Trap) linear gradient (3–80% acetonitrile in 0.1% formic acid). Then, the eluent was analyzed with a Q-TOF or Ion Trap mass spectrometer.