From Solution to the Gas Phase: Factors That Influence Kinetic Trapping of Substance P in the Gas Phase

Abstract

Substance P (RPKPQQFFGLM-NH2) [M + 3H](3+) ions have been shown to exist as two conformers: one that is kinetically trapped and one that is thermodynamically more stable and therefore energetically preferred. Molecular dynamics (MD) simulations suggested that the kinetically trapped population is stabilized by interactions between the charge sites and the polar side chains of glutamine (Q) located at positions 5 and 6 and phenylalanine (F) located at positions 7 and 8. Here, the individual contributions of these specific intramolecular interactions are systematically probed through site-directed alanine mutations of the native amino acid sequence. Ion mobility spectrometry data for the mutant peptide ions confirm that interactions between the charge sites and glutamine/phenylalanine (Q/F) side chains afford stabilization of the kinetically trapped ion population. In addition, experimental data for proline-to-alanine mutations at positions 2 and 4 clearly show that interactions involving the charge sites and the Q/F side chains are altered by the cis/trans orientations of the proline residues and that mutation of glycine to proline at position 9 supports results from MD simulations suggesting that the C-terminus also provides stabilization of the kinetically trapped conformation.

Cryogenic Ion Mobility-Mass Spectrometry: Tracking Ion Structure from Solution to the Gas Phase

“…26 Initial MD simulations of [SP + 3H] 3+ ions ( Figure 5) showed that compaction of the kinetically trapped species likely resulted from two predominant interaction types:…”

“…Substitution of alanine for native amino acids was used to eliminate potential interaction sites with minimal steric effects on ion structure. 26 Since the charge sites of SP were shown to interact primarily with glutamine and phenylalanine residues, a series of SP mutants were generated in which different combinations of Q 5 , Q 6 , F 7 , and F 8 were replaced by alanine. For example, the notation Q5,6A refers to the SP mutant ion in which glutamines in positions 5 and 6 have been replaced by alanines.…”

“…The increased Coulombic repulsion between highly localized charge sites that accompanied the loss of stabilizing interactions was shown to decrease the abundance of [SP M + 3H] 3+ ions, as previously noted, by leading to charge reduction reactions that occur via proton transfer from the peptide ion to surrounding water clusters. 27 26 Therefore, kinetic trapping of A is contingent on the formation of stabilizing intramolecular interactions between charge sites and key residues (Q 5 /Q 6 and F 7 /F 8 ), and disruption of these interactions leads not only to destabilization of the kinetically trapped species but also to the preferential formation of [SP + 2H] 2+ ions resulting from charge reduction reactions.…”

Penultimate Proline in Neuropeptides

“…12,20,21 The evaporative cooling process of ESI is thought to aid in kinetically trapping solution populations. [22][23][24] In a recent study, we reported that peptides with a proline residue located at the second position from the N-terminus (i.e., penultimate proline) have a propensity to adopt multiple conformations from the cis-trans isomerization of Xaa 1 -Pro 2 peptide bonds. 17 We subsequently noticed that penultimate proline residues are frequently located in many bioactive peptides, prompting us to inspect the structural implications of penultimate proline residues.…”

Toward a Rational Design of Highly Folded Peptide Cation Conformations. 3D Gas-Phase Ion Structures and Ion Mobility Characterization

“…Several previous studies of gas-phase peptide ions generated by electrospray used ion mobility measurements that yielded collision cross sections that are sensitive to ion structure [22, 23]. Recent studies by Russell et al showed that for some peptides, the ion conformers found in the gas phase depend on the electrospray solvent and interface temperature, leading to the formation of thermodynamically most stable conformers or those with “frozen” conformations pertinent to solution or electrospray droplets [24, 25]. Several gas-phase ions derived from natural [26–29] or modified peptides [30] have been found to correspond to the lowest free-energy conformers, as determined by a combination of collision cross sections from ion mobility measurements and vibrational band features from multiphoton infrared action spectroscopy.…”