4700 proteomics analyzer maldi tof tof mass spectrometer

Gel slices were cut into pieces, dehydrated with acetonitrile (ACN), and digested in sequence-grade trypsin (15 μl of 12.5mg/ml) in the presence of ammonia bicarbonate (15 μl of 50 mM solution) for 12-14 hours at 37°C. Water soluble and hydrophobic peptides were combined following extraction of the latter using formic acid (FA, 5%) in 60% ACN, vacuum-dried. The dried peptide mix was reconstituted with 0.5%TFA in 50% ACN and deposited on the MALDI plate and dried prior to adding 0.5 ml of a 1:1 dilution of saturated α-cyano-4-hydroxycinnamic acid (CHCA) in 0.5%TFA in 50% ACN. Samples were analyzed using an Applied Biosystems 4700 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer, and proteins identified by combined MS+MS/MS data using the MASCOT (v2.0.00) search engine in GPS Explorer 3.6 (Applied Biosystems). A peptide m/z tolerance of 0.5 Da, a fragment ion m/z tolerance of 0.2 Da, fixed carbamidomethyl modification on the cysteine and variable methionine oxidation, and one possible missed cleavage for trypsin was used as search criteria. A confidence index (C.I.) above 95%, and a MS/MS score of 21 or greater were used as the cutoffs for positive hits.