Structural characterization of peptides via tandem mass spectrometry of their dilithiated monocations

A series of singly charged dilithiated peptides [Pep − H + 2Li]+, has been prepared in situ by electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) and their collisionally activated dissociations (CAD) have been examined by quadrupole ion trap or quadrupole/time-of-flight tandem mass spectrometry, respectively. In the [Pep − H + 2Li]+ derivatives, an acidic proton of the peptide is replaced by a lithium cation, while the other lithium cation provides the charge. Upon CAD, the Li+ ions become mobile along the backbone of the peptide, inducing fragmentations that produce sequence ions. Density functional theory calculations carried out for the dipeptide complex [PheGly − H + 2Li]+ predict the existence of several isomers, including ions with lithium carboxylate and lithium amide structures with one or two salt bridges. Because the relative energies of these isomers are relatively small, all may be populated upon typical CAD experiments. This explains the presence of protonated, singly lithiated as well as dilithiated fragments in the CAD spectra of [Pep − H + 2Li]+. In general, the CAD spectra contain almost complete, structurally diagnostic yn**, cn** and an* ion series, which allow for definitive sequence determination (the number of * indicates the number of metal ions in the fragments).The formation of C- as well as N-terminal products from [Pep − H + 2Li]+ agrees well with both carboxylate and deprotonated amide structures being populated upon CAD, consistent with the theoretical prediction. Besides promoting sequence-specific fragmentations, dilithiation of the precursor ions also enables the elimination of radicals (at low collision energies) to form dilithiated peptide α-backbone radicals. Very similar characteristics are observed for singly charged disodiated peptides.