Tof tof 5800 system

2-D gel electrophoresis spots of interest were excised and transferred into 1.5 ml sterile LoBind tubes (Eppendorf) for in-gel protein digestion. Gel pieces were de-stained with 100 μl of 50% (vol/vol) ACN/50 mM ammonium bicarbonate (NH4HCO3) for 10 min at room temperature on tube-rotating mixers. De-staining was repeated for at least three times or until the gel pieces became colourless. Gel pieces were dehydrated in 100% acetonitrile and dried in a Concentrator plus vacuum centrifuge (Eppendorf). The dried gel pieces were rehydrated with 50 μl of a 1:40 dilution of trypsin (15 ng/μl, sequencing grade) in 50 mM NH4HCO3 and incubated at 4°C for 1 h. After digestion, excess trypsin was removed and 20 μl of 50 mM NH4HCO3 was added to cover the gel pieces. The tubes were then incubated at 37°C for 16 h. Final protein extraction was performed on ice by sonication as follows: 2 s (sonication) and 4 s (rest) for 20 min in total. The supernatant was then dried using a Concentrator plus vacuum centrifuge (Eppendorf) at 45°C for 20 min. MALDI TOF (matrix-assisted laser desorption ionization–time of flight) mass spectrometry was performed using a TOF/TOF 5800 system (AB SCIEX) for peptide mass fingerprinting and protein identification.

MALDI-TOF MS and TOF/TOF tandem MS/MS were performed on an AB SCIEX TOF/TOF™ 5800 System (AB SCIEX, Framingham, MA). MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 4000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 4000 laser shots per fragmentation spectrum on each of the 7–10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions).