Deducing disulfide patterns of cysteine-rich proteins using signature fragments produced by top-down mass spectrometry

Direct mapping of protein disulfide pattern using top-down mass spectrometry (MS) is often hampered by inadequate fragmentation at the disulfide-enclosing region{,} and insufficient structural information provided by the fragments. Here we used electron-transfer/high energy collision dissociation (EThcD) to improve the fragmentation efficiency{,} and developed strategies that minimize false positive identification of fragments and deconvolute signals representing specific modifications made to the disulfide-cleavage-induced fragments. We observed clear correlations between unique modification (attachment or removal of H or SH) pattern and the number of disulfide bonds that enclose the corresponding region. Using the characteristic signature fragments{,} we in part localized Cys-bridging sites in disulfide-scrambled lysozyme{,} and reduced the number of putative disulfide patterns from 104 to 6. The results demonstrated the feasibility of direct analysis of complex disulfide patterns using top-down MS.