4800 plus tof tof analyzer

CBB-stained bands corresponding to the bands detected by the anti-G50 antiserum were excised from the gel and treated by in-gel digestion with a proteomics-grade trypsin (Sigma-Aldrich, St Louis, MO, USA). The digested samples were purified by using ZipTip C18 (Merck). The purified peptides were mixed with an equal volume of α-cyano-4-hydroxycinnamic acid (4-CHCA) (Shimadzu GLC Ltd., Tokyo, Japan) matrix solution (prepared as 10 mg/mL in 50% acetonitrile containing 0.1% TFA), and an aliquot of the mixture (0.5 μL) was spotted onto an ABI 4800 MALDI plate (SCIEX, Framingham, MA, USA). The mass spectrometer, a MALDI-TOF/TOF mass spectrometer (4800 Plus TOF/TOF analyzer, SCIEX) was tuned and calibrated using a calibration standards mixture (Peptide Calibration Standard II, Bruker Daltonics GmbH & Co. KG, Bremen, Germany) prior to the measurements. After the spot had been allowed to dry at room temperature, MS and MS/MS spectra were obtained. Proteins were identified using a Mascot search engine (ver. 2.4.0.; Matrix Science, London, UK) and the NCBI database.