Tfa final concentration

Mass spectra were acquired in a positive-ion reflector mode with the use of a 4800 Plus MALDI-TOF/TOF Analyzer (Applied Biosystems, Framingham, USA). Alpha-cyano-4-hydroxycinnamic acid (CHCA) from Sigma-Aldrich (Munich, Germany), dissolved in 50:50 water/acetonitrile (J.T. Baker, Deventer, The Netherlands) with 0.1% TFA – final concentration (Sigma-Aldrich, Munich, Germany), was exploited as a MALDI matrix. External calibration was achieved with a 4700 proteomics analyzer calibration mixture provided by Applied Biosystems. Samples were spotted onto a 384 Opti-TOF MALDI plate and analyzed. Data Explorer Software, Version 4.9 was applied to process acquired spectra. Mascot Distiller Software (version 2.5.1.0, Matrix Science) was employed to predict fragment ions from given peptide sequences and overlay them on the acquired MS/MS spectra.

Molecular weight measurements were performed with 4800 MALDI-TOF/TOF Analyzer (Applied Biosystems, Framingham, USA). The mass spectrometer equipped with a 200 Hz, 355 nm Nd:YAG laser operated in positive ion reflector mode. The acceleration voltage was set to 20 kV, lens to 10 kV and the delayed extraction time to 250 ns. Each mass spectrum was obtained by the accumulation of 1024 laser shots. Alpha-Cyano-4-hydroxy-cinnamic acid (Sigma-Aldrich, Munich, Germany) dissolved in 50:50 water/acetonitrile (J.T. Baker, Deventer, The Netherlands) with 0.1% TFA – final concentration (Sigma-Aldrich, Munich, Germany) was the matrix used. The instrument was calibrated with a 4700 proteomics analyzer calibration mixture provided by Applied Biosystems. Acquired spectra were processed with Data Explorer Software, Version 4.9 (Applied Biosystems, Framingham, USA). Fragmentation spectra were performed for the selected precursor ions detected in the MS scan using air as the collision gas and a collision energy of 1 kV. The instrument was calibrated using the fragmentation spectrum of Glu1-Fibrinopeptide B (EGVNDNEEGFFSAR). The MS/MS spectra resulted from the accumulation of 2000 laser shots.