Ultraflex 2 tof tof mass spectrometer

The protein bands were excised from the gels and the slices incubated for 45 min at 50°C with 10 mM dithiothreitol (Sigma) in 100 mM ammonium bicarbonate. After removal of the solution, the slices were treated for 45 min with 55 mM iodoacetamide (Sigma) in 100 mM ammonium bicarbonate in the dark to modify cysteine residues. The gel pieces were then washed three times with water, twice with 50 mM ammonium bicarbonate and finally with 50 mM ammonium bicarbonate in 50% acetonitrile. After drying under a gentle stream of nitrogen, the pieces were re-swollen in 20 µL 50 mM ammonium bicarbonate (pH 8.0) and digested with trypsin or chymotrypsin (Promega) overnight at 37°C. The peptide mass fingerprint spectra were recorded on an Ultraflex-II TOF/TOF mass spectrometer (Bruker Daltonic) equipped with MALDI source, nitrogen laser, LIFT cell for fragment ion post-acceleration, and a gridless ion reflector. The software Flex Control 2.4, Flex Analysis 2.4, and Biotools 3.0 were used to operate the instrument and to analyze the data. For external calibration, a peptide calibration mixture (Bruker Daltonics) was used. The methylated peptides were fragmented using the LIFT method (Suckau et al. 2003 (link)). MALDI samples were prepared as follows. One microliter of a DHB-matrix (7 mg 2.5-dihydroxybenzoic acid in 100 µL methanol) was mixed with 1 µL digest and deposited on the target.