MALDI-TOF/TOF mass spectrometry

MALDI-TOF/TOF mass spectrometry was performed as described previously⁹. Briefly, a saturated solution of α-cyano-4-hydroxycinnamic acid (CHCA) (Sigma) in 50% acetonitrile/0.1% TFA (Fluka AG) was used as a MALDI matrix. The protein mixture was spotted onto the MALDI target plate and allowed to dry at 25 °C. MALDI-TOF/TOF and MS/MS mass spectra were obtained in the positive ionization mode using ABI 4700 and ABI 4800 Proteomics Analyzers (Applied Biosystems) equipped with a solid state laser (diode pumped Nd:YAG laser) pulsing at a repetition rate of 200 Hz (pulse duration < 500 ps) and operating at a wavelength of 355 nm. The spectra were acquired using a dual-stage reflectron mirror and accumulated from up to 2500 and 20,000 shots in MS and MS/MS mode, respectively. The instrument was calibrated externally using a mixture of five peptide standards. Accelerating voltages applied for MS and MS/MS measurements were 20 and 8 kV, respectively. In MS/MS mode, a collision energy of 1 kV was applied, and nitrogen was used as a collision gas in collision-induced dissociation experiments. Raw spectral data were further processed using DataExplorer 4.5 software (Applied Biosystems). Database searches were performed against non-redundant protein sequence databases (Uni-Prot, Trembl, MSDB and NCBI) using the program Mascot (Matrix Science Ltd.).