Stereochemical Sequence Ion Selectivity: Proline versus Pipecolic-acid-containing Protonated Peptides

Abstract

Substitution of proline by pipecolic acid, the six-membered ring congener of proline, results in vastly different tandem mass spectra. The well-known proline effect is eliminated and amide bond cleavage C-terminal to pipecolic acid dominates instead. Why do these two ostensibly similar residues produce dramatically differing spectra? Recent evidence indicates that the proton affinities of these residues are similar, so are unlikely to explain the result [Raulfs et al., J. Am. Soc. Mass Spectrom. 25, 1705-1715 (2014)]. An additional hypothesis based on increased flexibility was also advocated. Here, we provide a computational investigation of the "pipecolic acid effect," to test this and other hypotheses to determine if theory can shed additional light on this fascinating result. Our calculations provide evidence for both the increased flexibility of pipecolic-acid-containing peptides, and structural changes in the transition structures necessary to produce the sequence ions. The most striking computational finding is inversion of the stereochemistry of the transition structures leading to "proline effect"-type amide bond fragmentation between the proline/pipecolic acid-congeners: R (proline) to S (pipecolic acid). Additionally, our calculations predict substantial stabilization of the amide bond cleavage barriers for the pipecolic acid congeners by reduction in deleterious steric interactions and provide evidence for the importance of experimental energy regime in rationalizing the spectra. Graphical Abstract ᅟ.

Energetic switch of the proline effect in collision‐induced dissociation of singly and doubly protonated peptide Ala‐Ala‐Arg‐Pro‐Ala‐Ala

“…Relative energies of amide N protonated structures, transition states, and fragment proton‐bound dimers (PBD) were calculated and shown in Scheme S1 (Supporting Information). The Ala‐Pro amide N (Nc(S), in which c represents the third amide bond and S denotes the absolute configuration of the proline nitrogen atom) was indeed the energetically most favored amide nitrogen protonation site. While the b 3 ‐y 3 fragmentation pathway was energetically preferred, the difference between the energy barriers of different [AAAPAA+H] + dissociation pathways was less than 2.3 kcal/mol.…”

“…Breci and coworkers analyzed the fragmentation data of doubly charged peptides in database, and concluded that the side chain of the adjacent residue to Pro played a role in the cleavage of the Xxx‐Pro bond via restricting the conformation of the proline‐containing peptides . Recently, in the theoretical study of proline and pipecolic‐acid‐containing protonated peptides, Abutokaikah et al suggested that the stability of transition structures was directly affected by ring type and stereochemistry, and therefore differing the production distribution under low‐energy CID condition . Despite the progress above in understanding the proline effect, the mechanism of the N‐terminal adjacent basic residues suppressing the selective cleavage of proline remains to be elucidated.…”

“…2 Recently, in the theoretical study of proline and pipecolic-acidcontaining protonated peptides, Abutokaikah et al suggested that the stability of transition structures was directly affected by ring type and stereochemistry, and therefore differing the production distribution under low-energy CID condition. 19 Despite the progress above in understanding the proline effect, the mechanism of the N-terminal adjacent basic residues suppressing the selective cleavage of proline remains to be elucidated.…”

Mass spectrometry studies of the fragmentation patterns and mechanisms of protonated peptoids

“…This situation is similar to fragmenting proline-and/or pipecolic acidcontaining peptides. 61 Both proline and pipecolic acid contain a secondary amide bond, which is similar to the amide bonds in peptoids. Cleavage of the amide bond C-terminal to both residues will lead to a N-terminal oxazolone fragment with a fixed charge, and formation of the corresponding peptide y-ion will require transferring a non-mobile proton from the N-terminal fragment to the C-terminal fragment.…”

“…62 Both effects and related observations have been welldocumented in the literature. 57,[61][62][63][64][65][66] Our previous experiments using deuterium labeled peptoids suggest that the Y-ions contain a hydrogen atom abstracted from the C-Hα bond adjunction to the dissociating amide bond, which would correspond to mode A. An example of the formation of Y3 and Y4 from This article is protected by copyright.…”