Flexanalysis software v 3.0

Peptide samples after IMAC enrichment were analysed on a MALDI TOF mass spectrometer (Autoflex, Bruker Daltonics) operated in the reflectron positive ion mode previously calibrated with peptide calibration standard I (Bruker Daltonics) in the 500–5000 m/z range. The mass spectra were acquired, and the data were processed with Flexcontrol and Flexanalysis software (v. 3.0) (Bruker Daltonics, Bremen, Germany). Peptides were identified by Mascot server search against RIP2K sequence and using the following parameters: trypsin enzyme, missed cleavages: 3, modifications: methionine oxidation, cysteine carbamido-methylation, serine and threonine phosphorylation, singly protonated monoisotopic mass (M+H)¹⁺, peptide tolerance: 0.2 Da.

Mass spectra of trypsin digested proteins were obtained in Autoflex II MALDI TOF/TOF (Bruker Daltonics). Mass spectra were recorded in linear mode equipped with a pulsed N₂ laser (λ = 337nm, 50 Hz) at 54% power in positive ion mode. After MS spectra acquisition, the instrument was switched to LIFT mode. The MS/MS spectra of top ten peptides with highest intensity were recorded by fragmentation of these peptides using LID (laser induced dissociation). MS/MS spectra were acquired with a minimum of 4000 and a maximum of 8000 laser shots using the instrument calibration file. Spectra baseline subtraction, smoothing [26 (link)] and centroiding were performed in FlexAnalysis software v3.0 (Bruker Daltonics).