Preferential Cleavage of N-N Hydrazone Bonds for Sequencing Bis-arylhydrazone Conjugated Peptides by Electron Transfer Dissociation

Electron transfer dissociation (El D) was used to sequence bis-alylhydrazone (BAH)-cross-linked peptides through preferential cleavage of the hydrazone bond. On average, 58% of the observed ETD product ion abundance was accounted for by fragment ions due to selective cleavage of the N12-N13 hydrazone bond. Dissociation of the N12-N13 hydrazone bond yielded the two constituent peptides, one an even-electron product ion termed L alpha 12, the other an odd-electron radical ion termed L beta 11(center dot), which allowed each peptide to be individually sequenced by MS/MS methods and the site of cross-linking to be identified. The proposed pathway for the dissociation of the hydrazone bond involves transfer of the electron directly to the protonated hydrazone functionality and subsequent rearrangement to yield the L alpha 12 and L beta 11(center dot) products. Collision induced dissociation (CID) of the even-electron L alpha 12 product yielded a series of b- and y-type ions; CID of the odd-electron L beta 11(center dot) product resulted in a wide range of fragment ions including a-, b-, c-, y-, and z-type ions.