Sequencing of the thirteen structurally isomeric quartets of N‐terminal dipeptide motifs in peptides by collision‐induced dissociation

Abstract

N-terminal sequencing of protonated peptides is challenging, since each b(2) ion represents two sequence isomers, e.g., NE and EN. Additionally the occurrence of compositional isomers, such as NE and QD, further increases the number of isomers to four (NE, EN, QD, DQ). This leads to a subset of 13 b(2) ion masses where each value represents four individual species. The b(2) ions within such a quartet are characterized by the same elemental composition. To test the utility of CID for differentiation of isomeric b(2) ions, the CID spectra of 52 small synthetic peptides were recorded, representing the 13 isomeric b(2) ion quartets, which may be formed from unmodified amino acid residues. The CID spectra of protonated peptides containing these quartets were carefully inspected for N-terminal sequence information. Below the m/z value of the b(2) ion, individual differences were found in the b(2) fragment ion signatures (neutral loss of CO, H(2)O, NH(3), and other less common units). Recognition of N and Q in second position from the N-terminus is based on c(1) ion formation. Relative intensities of immonium ions were also used for differentiation between sequence isomers. In the complementary high-mass regions above the m/z value of the y(max-2) ion, individual differences were observed in the formation of y(max-1), x(max-1) and z(max-1) ions, which could be correlated to the complementary low-mass ions. In summary, de novo sequencing of the N-terminal dipeptide motif is feasible by considering all available sequence information present in CID spectra of protonated peptides.

Structural and mechanistic information on c1 ion formation in collision-induced fragmentation of peptides

“…This model accounts for the formation of c 1 ions, but is not able to explain variable signal intensities of the c 1 ion relative to the b 2 ion. In a recent study by our group, the presence of c 1 ions was observed upon fragmentation of peptides containing a Q or N residue in N-terminal position 2 for nine synthetic peptides [6]. We also observed that c 1 ions were constantly accompanied by their complementary high mass fragments, the z max-1 ions.…”

“…A set of pure compound synthetic peptides (starting with YQ) was used for this three cases, and that the differences in the intensities of the c 1 ion are not related to differing b 2 ion structures, but must have their origin in another process. Such a result indicates a significant impact of a second mechanism of c 1 ion formation as was deduced from the presence of z max-1 ions in an earlier study [6].…”

“…A c 1 ion of low intensity was observed for peptides with N in position 2. These fragmentation patterns indicate that the structure of the b 2 ions containing N might be diketopiperazines, as an oxazolone structure would result in different fragmentation patterns for inverted sequence motifs [6,7].…”

“…It is most likely that this ion is formed by direct fragmentation of the molecular ion without a b 2 ion intermediate. The difference in the signal intensity of the a 2 ion is presumably correlated with the more intense Y immonium ion which can be formed by secondary fragmentation of the a 2 ion [6]. To further investigate the differences in the fragmentation profiles of peptides containing N in their N-terminal dipeptide motif, the mass regions below the b 2 ions of our set of synthetic peptides were investigated.…”

“…The high abundance and stability of b 2 ions in combination with the virtual lack of b 1 ions is the most common example of a peptide terminus-specific fragmentation [5]. Due to this lack of fragmentation between the first two residues, comprehensive interpretation of low mass fragment ions in peptide CID spectra is required for unique sequence information about the N-terminal dipeptide motif, which otherwise is a blind region in peptide sequencing by CID [6,7]. Another less extensively studied terminal fragmentation process is the formation of c 1 ions, which so far has only been reported for selected N-terminal dipeptide motifs with glutamine in position 2 [8].…”