Abi 4800 plus maldi tof tof mass spectrometer

Protein spots of interest were cut out manually from the gel and put into siliconized microcentrifuge tubes. In-gel digestion, MALDI-TOF-MS, and database searching were performed by a commercial company (Shanghai Boyuan Biological Technology Co Ltd, Shanghai, China). The protein spots were analyzed with an ABI 4800 plus MALDI-TOF/TOF mass spectrometer (AB SCIEX, Les Ulis, France) after in-gel digestion. Peptide mass mapping was carried out using the Mascot V2.1 search engine (Matrix Science, London, United Kingdom) against the NCBI protein databases with a GPS Explorer software, V3.6 (Applied Biosystems, Waltham, Massachusetts). Proteins were successfully identified (Figure 1).

The eluted compounds were desalted with ZipTip C₁₈ tips (Millipore, Billerica, MA) for MALDIanalysis. The ZipTip C₁₈ protocol consists of several steps: Tip solvation with acetonitrile, conditioning with 0.1 % TFA (repeating this step several times), sample loading (by pipetting up and down several times), washing with 0.1 % TFA and final elution with 5 μL of α-CHCA (5 mg/mL in acetonitrile-water 70/30, 0.1 % TFA).
For MALDI analysis, 0.8 μL of the eluted sample was placed directly on the MALDI target until complete evaporation. Mass spectra were acquired with an ABI4800 Plus MALDI-TOF/TOF mass spectrometer (AB/Sciex, Concord, Ontario, Canada), obtained in reflector mode and all the experiments were carried out in positive ion mode. For MS/MS experiments, precursor ions were selected by means of a timed ion selector at a resolution value of 200 (full width at half maximum). The Metastable Suppressor lenses were activated in all the MS/MS experiments to avoid the detection of remaining precursor ions and unwanted metastable decay fragments [9 ].
Data Explorer software was used for the analysis of the spectra (including smoothing and noise filtering). Only monoisotopic m/z ratios are reported. The mass spectrometer was externally calibrated with a peptide mixture provided by the manufacturer.