2.4. Determination of the oxidation degree
Samples were subjected to enzymatic digestion with trypsin
and were subsequently desalted by using extraction solid phase
Cleanup C18 Pipette Tips with a gradient of acetonitrile in order
to fractionate the sample. The desalted peptide was mixed 1:1
with -cyano-4-hidroxicinnamico acid (CHCA), then applied onto
a MALDI plate and allowed to dry at room temperature. For
PMF analysis spectrometer was operated in the reflectron mode,
m/z range 700–3500, the laser power was 70 and 100 sample
profiles were acquired, where each profile was the average
of 20 laser shots. For calibration PeptideMass Calibration Kit
(Sigma, Cat. No-1KT MSCAL2) was used. The spectra were processed
with the program Axima Biotech Launchpad (Shimadzu