Mass spectrometric (MS) analysis of folded hV3 to map the oxidation state of cysteines. (A and B) Representative MS/MS profile of folded hV3 monomer obtained from in-gel trypsin digested samples prepared without iodoacetamide treatment followed by analysis on a MALDI-ToF/ToF mass spectrometer. The regions marked in red (residue numbers are indicated above each peak) are the peaks identified using BioTools v3.2 as corresponding to hV3. Note that hV3-generated peptides show lower ionization efficiency owing to the inherent hydrophobicity of the sequence. Peaks corresponding to the cysteine residues of hV3 have been presented in B and are marked by red arrows. None of the observed peaks could be mapped to a disulfide-bonded species. (C) Total coverage of the MS data on the hV3 sequence (marked in red). The cysteines have been highlighted in yellow. Four of the six cysteines were identified from the MS studies. The other cysteines (C36 and C122) are present in large hydrophobic segments. It has not been possible to map these peptides even in prior MS studies of hV3, owing to the extremely hydrophobic nature and poor ionization efficiency of these segments (Saletti et al., 2017). MS fingerprinting using other proteases such as pepsin also did not yield the peptide fragments corresponding to these cysteines (not depicted).