Peptide calibration standard 2

The solvents from HPLC fractions were evaporated (Speed vac® plus SC110A Savant) and the dried peptide extracts were first dissolved in 5 µl of an aqueous solution containing 0.1% trifluoroacetic acid (TFA) and 5% ACN. The samples were subsequently mixed (1:1) with the matrix solution, 5 mg/ml α-cyano-4-hydroxycinnamic acid (α-CHCA) in acetonitrile/water (50:50) containing 0.1% TFA. 1µl droplets were applied on the MALDI target plate and spectra were subsequently acquired in a Bruker UltrafleXtreme mass spectrometer equipped with a nitrogen laser (337 nm). The spectrometer was operated in the reflectron positive-ion mode, using delayed extraction. Mass range was 600-3500, laser attenuation was set at 40% and ca. 2000 laser shots were acquired. Full MS spectra were externally calibrated with Bruker Peptide Calibration Standard II. The mass spectrometer was operated with FlexControl 3.4 software, and data treatment was performed by the FlexAnalysis 3.4 software package.

The interesting spots in the 2-DE gels were manually excised and decolorized in a destainer (100 mmol/l NH 4 HCO 3 , 30% (v/v) acetonitrile, and double distilled H 2 O) for 0.5 h. Subsequently, the distained gel particles were dehydrated using 100% (v/v) acetonitrile twice, and then the dried gel particles were soaked with 10 ng/µl trypsin at 4°C for 60 min. Thereafter, the samples were digested in 25 mmol/l NH 4 HCO 3 (pH 8.0) for at least 20 h at 37°C. The collected supernatant peptide solution (1 µl) was spotted onto an Anchorchip target (Bruker-Daltonics, Bremen, Germany) and air-dried at room temperature for 15 min. After that, the same volume of a matrix (10 mg/ml α-cyano-4-hydroxy-trans-cinnamic acid, 50% (v/v) acetonitrile, and 0.1% (v/v) trifluoroacetic acid) was used to cover the dried peptide spots and air-dried at room temperature for another 15 min. Finally, MS analyses of the peptide spots were performed on an Ultraflex MALDI-TOF/TOF mass spectrometer (Bruker-Daltonics). All mass spectra were calibrated using the Bruker peptide calibration standard II (Bruker-Daltonics).

A Bruker UltrafleXtreme MALDI-TOF/TOF instrument (Bruker Daltonics) was used to acquire the MALDI-MS data using an optimized method. This method covered the mass range of m/z 80 to 600 in Reflectron positive mode, with laser power 31% on target. Data from a sum of 1000 laser shots were acquired in 100-shot steps, spiral_ small measuring raster movement, at every sample spot within the 48-by-32 spot target. The instrument was calibrated using Bruker Peptide Calibration Standard II and well-known peaks associated with the CHCA matrix (m/z 190.0 and m/z 379.1) to ensure calibration across the entire range. FlexControl software v3.4 (Bruker Daltonics) and FlexImaging software v4.0 (Bruker Daltonics) were used to control data acquisition and to generate the geometric spectra configuration for each target, respectively. Within the FlexImaging software, the spot microarray preparation mode was chosen to enable the Teach sample option; three Teach points (top left, top right, and bottom right sample spots) were set up to map the optical image coordinates to related positions on the sample carrier (MALDI MTP target), resulting in a rectangular shape. The number of spots was then specified in both the horizontal and vertical directions (e.g., x = 48 and y = 32).