Blackbody Infrared Radiative Dissociation of Bradykinin and Its Analogues:  Energetics, Dynamics, and Evidence for Salt-Bridge Structures in the Gas Phase

Abstract

Blackbody infrared radiative dissociation (BIRD) spectra of singly and doubly protonated bradykinin and its analogues are measured in a Fourier-transform mass spectrometer. Rate constants for dissociation are measured as a function of temperature with reaction delays up to 600 s. From these data, Arrhenius activation parameters in the zero-pressure limit are obtained. The activation parameters and dissociation products for the singly protonated ions are highly sensitive to small changes in ion structure. The Arrhenius activation energy (E a ) and pre-exponential (or frequency factor, A) of the singly protonated ions investigated here range from 0.6 to 1.4 eV and 10 5 to 10 12 s −1 , respectively. For bradykinin and its analogues differing by modification of the residues between the two arginine groups on either end of the molecule, the singly and doubly protonated ions have average activation energies of 1.2 and 0.8 eV, respectively, and average A values of 10 8 and 10 12 s −1 , respectively, i.e., the presence of a second charge reduces the activation energy by 0.4 eV and decreases the A value by a factor of 10 4 . This demonstrates that the presence of a second charge can dramatically influence the dissociation dynamics of these ions. The doubly protonated methyl ester of bradykinin has an E a of 0.82 eV, comparable to the value of 0.84 eV for bradykinin itself. However, this value is 0.21 ± 0.08 eV greater than that of singly protonated methyl ester of bradykinin, indicating that the Coulomb repulsion is not the most significant factor in the activation energy of this ion. Both singly and doubly protonated Lys-bradykinin ions have higher activation energies than the corresponding bradykinin ions indicating that the addition of a basic residue stabilizes these ions with respect to dissociation. Methylation of the carboxylic acid group of the C-terminus reduces the E a of bradykinin from 1.3 to 0.6 eV and the A factor from 1012 to 105 s −1 . This modification also dramatically changes the dissociation products. Similar results are observed for [Ala 6 ]-bradykinin and its methyl ester. These results, in combination with others presented here, provide experimental evidence that the most stable form of singly protonated bradykinin is a salt-bridge structure.

Fragmentation of doubly-protonated peptide ion populations labeled by H/D exchange with CD3OD

“…Presumably, these ion pairs can be formed simultaneously by the same mechanism if, when the amide bond cleaves, one ionizing proton is located on each of the newly formed b n and y m−n ions. Williams and co-workers have shown by blackbody infrared dissociation (BIRD) that the lowest energy fragmentation pathway for doublyprotonated bradykinin results in the formation of the b 2 /y 7 ion pair [29]. This is supported by molecular dynamics simulations which demonstrate the protonated N-terminal arginine is partially solvated by the backbone carbonyl oxygen of the proline residue in the second position, which likely contributes to formation of the b 2 and y 7 ions [29].…”

“…Later studies by Guevremont and co-workers using high-field asymmetric waveform ion mobility spectrometry (FAIMS) combined with H/D exchange detected four separate bradykinin populations, including one of very low abundance, for the doubly-protonated peptide [23]. In general, ion mobility and H/D exchange results support a compact, folded conformation for both singly-and doublyprotonated bradykinin in which the basic and acidic sites interact [10,16,29]. The existence of a salt-bridge, in which the carboxy terminus is deprotonated and both arginine residues are protonated, has been proposed for singly-and doubly-protonated bradykinin [27,28].…”

Dissociation of different conformations of ubiquitin ions

“…However, an understanding of the relationship between gas phase structure and solution conformation is still at a very primitive stage, in large part because detailed gas phase structural information is limited. In addition to chemical probes of gas phase ion structure [1][2][3][4][5][6][7][8][9][10][11][12][13][14], a number physical methods are being developed to examine gas-phase peptide and protein ion structure [15][16][17][18][19][20][21][22][23][24][25][26][27], including collisional activation to examine fragmentation pathways [18 -23, 25-27]. A few studies have suggested that ion conformation may influence fragmentation pathways [19,23,27,28].…”

Evaluation of ion mobility spectroscopy for determining charge-solvated versus salt-bridge structures of protonated trimers

“…A number of different methods can provide information about molecular structure in the gas phase, even complex systems, such as large synthetic or biological polymers. These methods include spectroscopy [1][2][3][4][5], hydrogen/deuterium exchange [6-12], dissociation [13][14][15][16][17][18], proton transfer reactivity [19][20][21][22], and ion mobility mass spectrometry . These methods have been used to obtain information about protein conformation and folding, DNA duplex structure, and in a wide variety of other interesting applications.…”

“…Zwitterions are destabilized in the gas phase compared with solution, but there is considerable evidence that these ionic interactions play a key role in the structure and reactivity of biomolecules in the gas phase [13,14,60,61]. Bowers and coworkers have used ion mobility mass spectrometry to examine the structure of cationized amino acids and related molecules.…”