The influence of cation and “alternative” amino acids on the fragmentation pathways of metal cationized and protonated peptides
Tandem mass spectrometry and collision-induced dissociation (CID) are the “workhorse” methods for protein identification in proteomics investigations. Recent studies have demonstrated significant differences in the CID spectra of Li⁺, Na⁺ and silver cationized peptides, particularly with respect to the preferred product ions. For example, the former produce primarily (bn+17+Li)⁺ while the latter preferentially generates (bn-1+Ag)⁺ species. To improve our understanding of peptide fragmentation in general, three separate studies were initiated. The objective of the first study was to determine the CID patterns for thallium (I) cationized peptides and compare them to those from Ag, Na, and protonated analogues. The goal was to determine whether thallium, which represents a monovalent cation of relative hardness that differs from that of the group I metals, would demonstrate reaction pathways similar to group (I) cations or Ag (I). CID results show that the tendency to produce (bn+17+Tl)⁺ or (bn-1+Tl)⁺ depended significantly on the peptide sequence. Also, the multi-stage CID of Tl⁺ cationized peptides fails in the determination of the peptide sequence. The second objective of this research was to determine the influence of a 4-aminomethylbenzoic acid (4AMBz) residue on the relative intensities of (b₃-1+cat)⁺ and (b₃+17+cat)⁺ fragment ions using tetrapeptides of the general formula A(4AMBz)AX and A(4AMBz)GX (where X = G, A, V). For Li⁺ and Na⁺ cationized versions of the peptides there was a significant increase in the intensity of (b₃-1+cat)⁺ for the peptides that contain the 4AMBz residue, and in some cases the complete elimination of the (b₃+17+cat)⁺ pathway. The influence of the 4AMBz residue is attributed to generation of a highly-conjugated oxazolinone species as (b₃-1+cat)⁺, which increases the stability of this product relative to the rival (b₃+17+cat)⁺ ion. This conclusion is supported by dissociation profiles, which suggest that the energetic requirements for generation of (b₃-1+cat)⁺ are significantly lower when the 4AMBz residue is positioned such that it should enhance formation of the conjugated oxazolinone. The objective of the third study was to determine the effect of the same residues on the formation of (b₃-1+cat)⁺ products from metal (Li⁺, Na⁺ and Ag⁺) cationized peptides. The larger amino acids suppress formation of b₃⁺ from protonated peptides with general sequence AAXG (where X=β-alanine, γ-aminobutyric acid or ε-aminocaproic acid), presumably due to the prohibitive effect of larger cyclic intermediates in the “oxazolone” pathway. However, abundant (b₃-1+cat)⁺ products are generated from metal cationized versions of AAXG. Using a group of deuterium-labeled and exchanged peptides, we found that formation of (b₃-1+cat)⁺ involves transfer of either amide or α-carbon position H atoms, and the tendency to transfer the atom from the α-carbon position increases with the size of the amino acid in position X. To account for the transfer of the H atom, a mechanism involving formation of a ketene product as (b₃-1+cat)⁺ is proposed.
Thesis [M.S.] - Wichita State University, College of Liberal Arts and Science, Dept. of Chemistry