Influence of a Gamma Amino Acid on the Structures and Reactivity of Peptide a(3) Ions

Abstract

Collision-induced dissociation of protonated AGabaAIG (where Gaba is gamma-amino butyric acid, NH(2)-(CH(2))(3)-COOH) leads to an unusually stable a(3) ion. Tandem mass spectrometry and theory are used here to probe the enhanced stability of this fragment, whose counterpart is not usually observed in CID of protonated peptides containing only alpha amino acids. Experiments are carried out on the unlabelled and (15)N-Ala labeled AGabaAIG (labeled separately at residue one or three) probing the b(3), a(3), a(3)-NH(3) (a(3) (*)), and b(2) fragments while theory is used to characterize the most stable b(3), a(3), and b(2) structures and the formation and dissociation of the a(3) ion. Our results indicate the AGabaA oxazolone b(3) isomer undergoes head-to-tail macrocyclization and subsequent ring opening to form the GabaAA sequence isomer while this chemistry is energetically disfavored for the AAA sequence. The AGabaA a(3) fragment also undergoes macrocyclization and rearrangement to form the rearranged imine-amide isomer while this reaction is energetically disfavored for the AAA sequence. The barriers to dissociation of the AGabaA a(3) ion via the a(3)→b(2) and a(3)→a(3)* channels are higher than the literature values reported for the AAA sequence. These two effects provide a clear explanation for the enhanced stability of the AGabaA a(3) ion.

Figure 1

Product ion mass spectra of protonated (a) AGabaAIG, (b) GabaAAIG, and (c) AAAIG recorded on the G2 Synapt instrument at 22 eV collision energy (laboratory frame).

Figure 2

MS³ tandem mass spectra (recorded using SORI-CID after source fragmentation) of a) the b₂, b) the a₃*, c) the a₃ and d) the b₃ fragment ions of protonated AGabaAIG.

Figure 3

MS³ tandem mass spectra (recorded using SORI-CID) of (a) the b₃ of protonated (¹⁵N-A)GabaAIG and (b) the b₃ of protonated AGaba(¹⁵N-A)IG. Insets in (b) are expanded ranges of the m/z 140 and m/z 180-187 regions.

Figure 4

MS³ tandem mass spectra (recorded using SORI-CID) of m/z 201.13 (a) the a₃ of protonated (¹⁵N-A)GabaAIG and (b) the a₃ of protonated AGaba(¹⁵N-A)IG.

Figure 5

MS³ tandem mass spectra (recorded using SORI-CID) of the m/z 157.1 fragment of a) protonated (¹⁵N-A)GabaAIG and b) protonated AGaba(¹⁵N-A)IG.

Chart 1

Various structures for the b₃ fragment of protonated AGabaAIG: A) linear AGabaA isomer terminated by the oxazolone ring; B) protonated cyclo-(AGabaA); C) linear GabaAA isomer terminated by the oxazolone ring; D) linear AAGaba isomer terminated by a seven-membered ring. Characterized protonation sites are indicated by arrows with the corresponding relative energies (kcal/mol) and relative Gibbs free energies (in parentheses, kcal/mol). The relative energies are calculated with respect to the oxazolone protonated AGabaA_ox species A.

Chart 2

Structures for the b₂ fragment of protonated AGabaAIG and GabaAAIG: A) linear AGaba isomer terminated by the seven-membered ring; B) protonated eight-membered cyclo-(AGaba); C) linear GabaA isomer terminated by the oxazolone ring. As with previous charts, protonation sites are indicated by arrows with the corresponding relative energies (kcal/mol) and relative Gibbs free energies (in parentheses, kcal/mol). The relative energies are calculated with respect to the ring nitrogen protonated AGaba species.

Chart 3

Various a₃ (AGabaA sequence) ion structures: A & B) linear form protonated at the imine or N-terminal amino groups, respectively; C and D) macrocyclic (11-membered ring) isomer protonated at the secondary amine or the adjacent amide nitrogen, respectively; E) and F) rearranged linear form protonated at the N-terminal imine group or the C-terminal amide nitrogen, respectively; G and H) nine-membered ring isomer protonated at the N-terminal amine or ring imine nitrogen, respectively; I, J, and K) seven-membered ring terminated re-associated linear form protonated at the secondary amine, N-terminal amine, or ring nitrogen, respectively. The respective protonation sites are indicated by arrows. Relative ZPE-corrected energies (Gibbs free energies at 298 K) are given.

Scheme 1

Possible fragmentation pathways for formation of a₃ and its subsequent fragments proposed in the literature [14, 18, 22, 24].

Scheme 2

Fragmentation pathways of protonated AGabaA b₃ ion.

Scheme 3

Fragmentation pathways of AGabaA a₃ ion. Relevant transition state barriers reported relative to the a₃ structure A ion in kcal/mol.