The ornithine effect in peptide cation dissociation

Abstract

Facile cleavage C-terminal to ornithine residues in gas phase peptides has been observed and termed the ornithine effect. Peptides containing internal or C-terminal ornithine residues, which are formed from deguanidination of arginine in solution, were fragmented to produce either a y-ion or water loss, respectively, and the complementary b-ion. The fragmentation patterns of several peptides containing arginine were compared to those of the ornithine analogues. Conversion of arginine to ornithine results in a decrease of the gas phase proton affinity of the residue, thereby increasing the mobility of the ionizing proton. This alteration allows the nucleophilic amine to facilitate a neighboring group reaction to induce a cleavage of the adjacent amide bond. The selective cleavage at the ornithine residue is proposed to result from the highly favorable generation of a six-membered lactam ring. The ornithine effect was compared with the well-known proline and aspartic acid effects in peptide fragmentation using angiotensin II, DRVYIHPF and the ornithine analogue, DOVYIHPF. Under conditions favorable to either the aspartic acid (i.e. singly protonated peptide) or proline effect (i.e. doubly protonated peptide), the ornithine effect was consistently observed to be the more favorable fragmentation pathway. The highly selective nature of the ornithine effect opens up the possibility for conversion of arginine to ornithine residues to induce selective cleavages in polypeptide ions. Such an approach may complement strategies that seek to generate non-selective cleavages of the related peptides.

Keywords: deguanidination; lactam formation; ornithine; selective cleavage.

Figure 1

Comparison of the fragmentation patterns between arginine-and ornithine-containing peptides. (a) CID of [AAAAARA+H]⁺ produces several sequence fragments, neutral losses, and unidentifiable peaks. (b) CID of [AAAAAOA + H]⁺ produces a major cleavage C-terminal to ornithine. Ammonia loss is labeled with an asterisk (*), water loss is labeled with a degree sign (°). Precursor ion fragmented (M) is denoted with a lightning bolt ( ).

Figure 2

Comparisons of CID behavior of small peptides with basic C-terminal residues. (a) CID of [YGGFLR + H]⁺; (b) CID of [YGGFLK + H]⁺; (c) CID of [YGGFLO + H]⁺. Ammonia loss is labeled with an asterisk (*), water loss is labeled with a degree sign (°). Precursor ion fragmented (M) is denoted with a lightning bolt ( ).

Figure 3

Comparisons of the proline effect between arginine- and ornithine-containing peptides. (a) CID of [GAILPGAILR +H]⁺; (b) CID of [GAILPGAILO + H]⁺; (c) CID of [GAILPGAILO+ H-H₂O]⁺. Ammonia loss is labeled with an asterisk (*), water loss is labeled with a degree sign (°). Precursor ion fragmented (M) is denoted with a lightning bolt ( ).

Figure 4

Comparisons of the aspartic acid effect with arginine- and ornithine-containing peptides. (a) CID of [GAILDGAILR+ H]⁺;(b) 25× zoom in of CID of [GAILDGAILR + H]⁺ from top; (c) CID of [GAILDGAILO+ H]⁺. Ammonia loss is labeled with an asterisk (*), water loss is labeled with a degree sign (°). Precursor ion fragmented (M) is denoted with a lightning bolt ( ).

Figure 5

Comparison of CID of Angiotensin II (human), DRVYIHPF and ornithinated DOVYIHPF. (a) CID of [DRVYIHPF+H]⁺; (b) CID of [DOVYIHPF + H]⁺; (c) CID of [DRVYIHPF + 2H]⁺; (d) CID of [DOVYIHPF + 2 H]⁺. Water loss is labeled with a degree sign (°). Precursor ion fragmented (M) is denoted with a lightning bolt ( ).

Scheme 1

Conversion of arginine to ornithine.

Scheme 2

(Left) Nucleophilic attack of the carbonyl carbon by the amine results in the formation of (Right) a six-membered lactam and cleavage at the C-terminal carbonyl carbon bond of ornithine. If ornithine is N-terminal or internal, HR is an amine (HNH-R); if ornithine is the C-terminal residue, HR is water loss (H₂O).