Cyano 4 hydroxy cinnamic acid chca

For peptide mass fingerprinting (PMF), the spots were excised from the stained gel and in-gel digestion was carried out. The excised spots were destained with 50 mM ammonium bicarbonate in 40% acetonitrile, and dried with a speed vac (Heto Lab Equipment, Allerod, Denmark). The destained spots were rehydrated in 12.5 ng/μL trypsin in 50 mM ammonium bicarbonate. The rehydrated spots were placed on ice for 45 min and treated with 50 mM ammonium bicarbonate (10 μL). The spots were then incubated at 37 °C for 12 h.
For MALDI-TOF-MS analysis, the peptides were desalted and concentrated using a POROS R2 and Oligo R3 column (Applied Biosystems, Fostercity, CA, USA). The column was washed sequentially with 70% acetonitrile, 100% acetonitrile, and 50 mM ammonium bicarbonate. The samples were applied and eluted with cyano-4-hydroxycinnamic acid (CHCA) (Sigma, St. Louis, MO, USA) dissolved in 70% acetonitrile and 2% formic acid onto the MALDI plate (Opti-TOF™ 384-well Insert, Applied Biosystems) [35 (link)]. MALDI-TOF-MS was performed on a 4800 MALDI-TOF/TOF™ Analyzer (Applied Biosystems) equipped with a 355-nm Nd:YAG laser (TOF analyzer pressure: approximately 7.6 × 10⁻⁷ Torr). The mass spectra were obtained in the reflection mode (accelerating voltage of 20 kV and the sum from either 500 laser pulses), and calibrated using the 4700 calibration mixture (Applied Biosystems).