During biosynthesis of amphotericin, the macrolactone core undergoes three modifications: oxidation of a methyl branch to a carboxyl group, mycosaminylation, and hydroxylation.
The MASCOT search engine (version 2.4) was used for peptide sequence searching against the UniProt database, setting the following restrictions: Homo sapiens taxonomy (Human), parent ion tolerance ±20 ppm, MS/MS error tolerance ±0.1 Da, alkylation of cysteine residues (fixed modifications), oxidation of methionine (variable modifications), and two potentially missed trypsin cleavages.
The database searches were constrained by allowing for trypsin cleavage (with up to two missed cleavage sites), fixed modifications (carbamidomethylation of Cys residues) and variable modifications (oxidation of Met residues and N-terminal pyroglutamate formation).
The following parameters were used for searches using SEQUEST v.3.3.1 SP1: enzyme, trypsin; fixed modifications, carboxymethyl cysteine; variable modifications, oxidation of methionine; peptide tolerance, ±1.50 amu; fragment ion tolerance, ±0.5 amu; and number of missed cleavage sites, 1.
Precursor mass accuracy (10 ppm), MSMS mass accuracy (0.6 Da), enzyme (Trypsin, missed cleavages = 1), Fixed modifications (Carbamidomethylation of cysteine), Variable modifications (Oxidation of methionine, phosphorylation of S, T, Y), minimum ion score = 20.
MALDIM-MS ion search was performed with this set of parameters: (I) taxonomy: Mus musculus, (II) proteolytic enzyme: trypsin, (III) maximum of accepted missed cleavages: 1, (IV) mass value: monoisotopic, (V) peptide mass tolerance 0.8 Da, (VI) fragment mass tolerance: 0.8 Da, and (VII) variable modifications: oxidation of methionine and acrylamide adducts (propionamide) on cysteine.
In contrast to lignin modification, oxidation of cellulose impaired enzymatic hydrolysis.
As fixed modification, carbamidomethylation of cysteine residues was used, and as variable modification oxidation of methionine residues was selected.
ROS damages DNA by causing strand breakage, base modification, oxidation of deoxyribose, and DNA-protein cross-links [ 114].
Search settings allowed one missed cleavage with the trypsin enzyme selected, one fixed modification (carboxymethylated cysteine) and one variable modification (oxidation of methionine).
Parent and fragment mass tolerances were, respectively, set to 20 p.p.m. and 0.3 Da, partial modification (oxidation) of methionine residues being allowed.
