800 μm anchorchip

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.

Manually excised Coomassie stained gel spots were washed and digested according previously reported studies (20 (link), 21 (link)). The mixture of tryptic peptides (0.8 μL) derived from each protein was spotted onto a MALDI target (384 MTP Anchorchip; 800 μm Anchorchip; Bruker Daltonics, Bremen, Germany). MALDI-MS(/MS). As previously mentioned, spectra were obtained using an UltraflexTerm time-of-flight (TOF) mass spectrometer equipped with a LIFT-MS/MS device (Bruker Daltonics) at 21 kV reflector and 17 kV detector voltages, respectively (20 (link), 21 (link)). PMFs were calibrated against a standard (peptide calibration standard II, Bruker Daltonics). Flex Analysis software was used to assess the PMFs (Bruker Daltonics v.2.4). Interpretation of MS data was done using BioTools v3.2 (Bruker Daltonics). The peptide masses were searched against the Mascot search algorithm (v2.0.04, updated on 09/05/2021; Matrix Science Ltd., UK). Identified proteins were accepted as correct if they the Mascot score > 56. Because some proteins were in low abundance and did not give sufficiently powerful mass fingerprints, not all spots of interest could be recognized; some spots were mixtures of multiple proteins (30 (link), 31 (link)).