Ppsq 31b

N-Terminal sequence determination was performed by stepwise Edman degradation using an automatic sequencer model PPSQ 31B (Shimadzu, Kyoto, Japan).

MALDI in-source decay was performed using an Ultraflextreme TOF/TOF mass spectrometer controlled by the FlexControl 3.3 software (BrukerDaltonics). Spectra were acquired in positive reflectron ion mode with 2000 laser shots accumulated, and the laser power was set with an increase in 20% of fluensce to fragment protein in the source of the mass spectrometer using a 20 mg/mL solution of 1,8-diaminonaphtalen (DAN) matrix enabling the generation of hydrogen radicals that break the peptide backbone producing c-ions and z-ions from 1000 to 5000 Da. The generated fragment ions allowed the sequence annotation of the peptides. The mass spectrometer parameters were set according to the manufacturer’s settings for optimal acquisition performance. Sequences were analyzed on Flex Analysis software, version 3.0 (BrukerDaltonics). Mass spectrometer calibration was performed using the c-ions of 1 pmole of BSA spotted with the DAN matrix.
N-terminal sequencing of the peptide present on the active fraction was performed by Edman degradation. Approximately 600 pmol of the sample was loaded on a glass fiber membrane pretreated with 10 µL of biobrene and then analyzed using an automatic sequencer model PPSQ 31B (Shimadzu, Kyoto, Japan). The sequence was compared with the Uniprot database and with the sequence obtained by MS.