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

Abstract

Suppression of the selective cleavage at N‐terminal of proline is observed in the peptide cleavage by proteolytic enzyme trypsin and in the fragment ion mass spectra of peptides containing Arg‐Pro sequence. An insight into the fragmentation mechanism of the influence of arginine residue on the proline effect can help in prediction of mass spectra and in protein structure analysis. In this work, collision‐induced dissociation spectra of singly and doubly charged peptide AARPAA were studied by ESI MS/MS and theoretical calculation methods. The proline effect was evaluated by comparing the experimental ratio of fragments originated from cleavage of different amide bonds. The results revealed that the backbone amide bond cleavage was selected by the energy barrier height of the fragmentation pathway although the strong proton affinity of the Arg side chain affected the stereostructure of the peptide and the dissociation mechanism. The thermodynamic stability of the fragment ions played a secondary role in the abundance ratio of fragments generated via different pathways. Fragmentation studies of protonated peptide AACitPAA supported the energy‐dependent hypothesis. The results provide an explanation to the long‐term arguments between the steric conflict and the proton mobility mechanisms of proline effect.

Targeting proline in (phospho)proteomics

“…Because of its unique properties, proline often leads to missed proteolytic events during digestion [9], increasing the resultant peptide length and database search complexity. Moreover, proline also effects the fragmentation step during mass analysis, known as the 'proline effect' [20], where fragmentation shows enhanced production of y-ions spanning from the proline to the peptide C terminus due to the enhanced basicity of the proline nitrogen, restricting the peptide sequence coverage [20][21][22].…”

Mass spectral and theoretical investigations of the transient proton-bound dimers on the cleavage processes of the peptide GHK and its analogues

“…S1 in ESI. † The structure with the lowest energy was selected to perform a geometric optimization and to search reaction pathways 18 at the B3LYP/def-TZVP level using Turbomole supported by ChemShell 3.7.0. 34 Key transition states with only one imaginary frequency were further validated by intrinsic reaction coordinate (IRC) calculations to confirm their connections.…”

“…16 Several studies have suggested that the oxazolone structure is likely a b 2 isomer. [17][18][19][20] Moreover, the possibility for an isomerization between an oxazolone and a diketopiperazine form has been exploited. 21 In the diketopiperazine pathway, the N-terminal amino group acts as a nucleophile and attacks the carbonyl carbon of the second amino acid residue, forming a more stable six-membered ring structure (Path c in Scheme 1).…”