Amplified histidine effect in electron-transfer dissociation of histidine-rich peptides from histatin 5

We report electron-transfer dissociation (ETD) mass spectra of histidine-containing peptides DSHAK, FHEK, HHGYK, and HHSHR from trypsinolysis of histatin 5. ETD of both doubly and triply protonated peptides provided sequence ions of the c and z type. In addition, electron transfer to doubly protonated peptides produced abundant long-lived cation-radicals, (M+2H)+, whose relative intensities depended on the peptide sequence and number of histidine residues. CID-MS3 spectra of (M+2H)+ cation-radicals were entirely different from the ETD spectra of the doubly charged ions and involved radical-driven losses of C4H6N2 neutral fragments from the histidine residues and charge-driven backbone cleavages forming b and y ions. Product ions from CID of (M+2H)+ were further characterized by CID-MS4 spectra to distinguish the histidine residues undergoing loss of C4H6N2. The ETD-CID-MSn mass spectra are interpreted by considering radical-induced rearrangements of histidine side chains in the long-lived charge-reduced ions.

Figure optionsDownload high-quality image (128 K)Download as PowerPoint slideResearch highlights▶ ETD of histidine-rich peptides provides complete sequence coverage. ▶ Charge-reduced survivor ions undergo specific rearrangements. ▶ Multi-stage CID reveals the histidine rearrangement sites.