Partial Reduction and Two-Step Modification of Proteins for Identification of Disulfide Bonds

An experimental protocol was established to combine
partial reduction, cyanylation, and a second modification
step for the assignment of disulfide bonds in proteins that
are resistant to proteolysis under native conditions. After
proteolysis, disulfide bonds were assigned via MALDI
mass spectrometry with subsequent semiautomatic interpretation using the program SearchXLinks, which
enumerates all possible combinations of proteolytic fragments for all observed monoisotopic masses. The putative
assignment of disulfide bonds was confirmed by ISD and
PSD fragmentation of the corresponding protonated molecules.