Cyano 4 hydroxycinamic acid

Protein spots were excised from gels with a sterile scalpel and placed into Eppendorf tubes. Proteins were digested with trypsin (Promega, Madison, WI) as previously described [11 (link)]. For matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis, peptides were concentrated using a POROS R2, Oligo R3 column (Applied Biosystems, Foster City, CA). After washing the column with 70% acetonitrile, 100% acetonitrile, and 50 mM ammonium bicarbonate, samples were applied to the R2, R3 column and eluted with cyano-4-hydroxycinamic acid (Sigma, St. Louis, MO) in 70% acetonitrile and 2% formic acid onto the MALDI plate (Opti-TOF 384-well Insert, Applied Biosystems) [12 (link)]. MALDI-TOF MS analysis was performed on a 4800 MALDI-TOF/TOF Analyzer (Applied Biosystems) equipped with a 355-nm Nd:YAG laser. The pressure in the TOF analyzer was approximately 0.00010132 Pa. Mass spectra were obtained in reflectron mode with an accelerating voltage of 20 kV and sum of 500 laser pulses, and calibrated using the 4700 calibration mixture (Applied Biosystems). Data Explorer 4.4 (PerSeptive Biosystems, Framingham, MA) was used for monoisotopic mass data acquisition and extraction.