*4.11. Protein MS-MS Analysis*

Peptides were subjected to de novo sequencing based on matrix-assisted laser desorption ionization time of flight (MALDI-TOF/TOF) mass spectrometry (MS) and subsequent computational analysis at the Proteomics Centre of the Institute of Biochemistry, Vilnius University (Vilnius, Lithuania). The sample was purified as described previously in Materials and Methods. Tryptic digests (0.5 μL) were transferred on 384-well MALDI plate with 0.5 μL 4 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) matrix in 50% acetonitrile with 0.1% trifluoroacetic acid and analyzed with an Applied Biosystems/MDS SCIEX 4800 MALDI TOF/TOF™ mass spectrometer. Spectra were acquired in the positive reflector mode between 800 and 4000 *m*/*z* with fixed laser intensity at 3700 (Laser shots: 400; Mass accuracy: ±50 ppm). The most intense peaks of each survey scan (MS) were fragmented for

sequence analysis (Collision energy: 1 keV; CID: no CID or medium air pressure CID used; Laser intensity: 4200–4400; Laser shots: 500–1000; Fragment mass accuracy: ±0.1 Da). Sequence analysis and peak lists were generated using GPS Explorer™ De Novo Explorer.
