  Phosphorylation is one of the most important protein modifications, and
electron-transfer dissociation tandem mass spectrometry (ETD-MS/MS) is a
potentially useful method for the sequencing of phosphopeptides, including
determination of the phosphorylation site. Notably, ETD-MS/MS typically
provides useful information when the precursor contains more than three
positive charges. It is not yet used as an analysis method for large-scale
phosphopeptide production due to difficulties occurring in the production of
acidic phosphopeptides having more than three positive charges. To increase the
charge state of phosphopeptides, we used dinuclear metal complexes, which
selectively bind to the phosphate group in phosphopeptides with the addition of
positive charge(s). Dinuclear copper, zinc, and gallium complexes were tested
and it was found that the type of metal present in the complex strongly
affected the affinity of the phosphorylated compounds and their ETD
fragmentation. The dinuclear copper complex interacted weakly with the
phosphate groups and ETD-induced peptide fragmentation was largely suppressed
by the presence of Cu2+, which worked as an electron trap. The dinuclear
gallium complex was strongly bound to a phosphate group. However, the ligand
binding to gallium acted as an electron trap and the presence of dinuclear
gallium complex in the precursor for ETD-MS/MS hampered the sequencing of the
phosphopeptides, as in the case of dinuclear copper complexes. In contrast,
dinuclear zinc complexes efficiently bind to phosphopeptides with an increase
in the charge state, facilitating phosphopeptide sequencing by ETD-MS/MS. The
fragmentation of the ligand and peptide backbone in the dinuclear
zinc--phosphopeptide complex were competitively induced by ETD. These processes
are influenced by the ligand structure and so the detailed ETD fragmentation
pathways were investigated using density functional theory calculations.
