Mascot 2.4.1 search engine

Each sample was mixed with matrix solution containing α-cyano-4-hydroxycinnamic acid (0.3 g/L CHCA in a solution containing 2:1 ethanol:acetone, v/v) at the ratio of 1:10. A total amount of 1 µL of the mixture containing sample/matrix solution was spotted onto the MALDI plate (AnchorChip 800 μm, Bruker Daltonics, Bremen, Germany) and kept at room temperature to allow crystallization to occur. UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA) was used to perform MS analyses in the reflector mode in the m/z range of 700–3500 Da. The MS spectra were externally calibrated with the mixture of Peptide Calibration Standard and Protein Calibration Standard I (Bruker Daltonics, Billerica, MA, USA) at the ratio of 1:5. FlexControl 3.4 software (Bruker Daltonics, Billerica, MA, USA) was applied to acquire and process spectra. FlexAnalysis 3.4 (Bruker Daltonics, Billerica, MA, USA) was applied to perform protein database searches. Proteins were identified using the Mascot 2.4.1 search engine (Matrix Science, London, UK). The following search parameters were applied: Enzyme: trypsin; Fixed modifications: Carbamidomethylation on cysteine; Variable modifications: Oxidation on methionine; Protein mass: Unrestricted; Peptide mass tolerance: ±50 ppm; Maximum missed cleavage: 2.

Each sample was mixed with matrix solution containing α-cyano-4-hydroxycinnamic acid (0.3 g/L CHCA in a solution containing 2:1 ethanol/acetone, v/v) at the ratio of 1:10. A total amount of 1 µL of the mixture containing sample/matrix solution was spotted onto the MALDI plate (AnchorChip 800 μm, Bruker Daltonics, Bremen, Germany) and kept at room temperature to allow crystallization to occur. An UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA) was used to perform MS analyses in the reflector mode in the m/z range of 700–3500 Da. The MS spectra were externally calibrated with the mixture of Peptide Calibration Standard and Protein Calibration Standard I (Bruker Daltonics, Billerica, MA, USA) at the ratio of 1:5. FlexControl 3.4 software (Bruker Daltonics, Billerica, MA, USA) was applied to acquire and process spectra. FlexAnalysis 3.4 (Bruker Daltonics, Billerica, MA, USA) was applied to perform protein database searches. Proteins were identified using the Mascot 2.4.1 search engine (Matrix Science, London, UK). The following search parameters were applied: enzyme: trypsin; fixed modifications: carbamidomethylation on cysteine; variable modifications: oxidation on methionine; protein mass: unrestricted; peptide mass tolerance: ±50 ppm; maximum missed cleavage: 2.