Zwitterionic States in Gas‐Phase Polypeptide Ions Revealed by 157‐nm Ultra‐Violet Photodissociation

Abstract. Significant effort is being employed to utilize the inherent speed and sensitivity of mass spectrometry for rapid structural determination of proteins; however, a thorough understanding of factors influencing the transition from solution to gas phase is critical for correct interpretation of the results from such experiments. It was previously shown that combined use of action excitation energy transfer (EET) and simulated annealing can reveal detailed structural information about gaseous peptide ions. Herein, we utilize this method to study microsolvation of charged groups by retention of 18-crown-6 (18C6) in the gas phase. In the case of GTP (CEGNVRVSRE LAGHTGY), solvation of the 2+ charge state leads to reduced EET, whereas the opposite result is obtained for the 3+ ion. For the mini-protein CTrpcage, solvation by 18C6 leads to dramatic increase in EET for the 3+ ion. Examination of structural details probed by molecular dynamics calculations illustrate that solvation by 18C6 alleviates the tendency of charged side chains to seek intramolecular solvation, potentially preserving native-like structures in the gas phase. These results suggest that microsolvation may be an important tool for facilitating examination of native-like protein structures in gas phase experiments.

Section: C-trpcagementioning

confidence: 57%

Structural Effects of Solvation by 18-Crown-6 on Gaseous Peptides and TrpCage after Electrospray Ionization

Bonner

Hendricks

Julian

2016

“…The 828 peak may have a small contribution from M-45 (loss of COOH from the precursor). Zubarev and coworkers have interpreted M-44 and M-45 fragments as indicative of whether the C-terminus is deprotonated by a zwitterionic structure [23]. While many of the same high-energy fragments are present when the peptide is dissociated with 157 nm light in the ion trap (Figure 4b), the x 9 and x 8 peaks are particularly dominant.…”

Section: Resultsmentioning

confidence: 99%

“…We suggested that the long observation timescale on this instrument makes additional fragments observable [20]. Zubarev and coworkers have also applied this technique to cleave disulfide bonds [21] and investigate peptide zwitterions in the gas-phase [22,23]. Morgan and Russell have compared 193 nm photodissociation in a tandem-TOF to their prior work using stepped reflectron voltages, and have studied some peptides with C-terminal arginine.…”

mentioning

confidence: 99%

Factors that impact the vacuum ultraviolet photofragmentation of peptide ions

Thompson

Cui

Reilly

2007

101

Several groups have investigated the photodissociation of peptide ions with ultraviolet light. Significant differences have been reported with 157 and 193 nm excitation. Recent studies have shown that the mass analyzer can also influence the observed photofragment distribution.Comparison of experiments using different peptides, wavelengths, and mass analyzers is undesirably complicated. In the present work, several peptides are analyzed with both 157 and 193 nm photodissociation in tandem-TOF and linear ion trap mass spectrometers. The results indicate that the fragment ion distribution can be influenced by both the photodissociation wavelength and the mass analyzer. The two wavelengths generate similar spectra in an ion trap but quite different results in a tandem

“…The fit parameters in Table 4 indicate similar values for all species and charge states except the wild Trp-cage sequence for 1ϩ, suggesting that a salt bridge plays a role in only this species. These results are particularly interesting in light of ultraviolet photodissociation measurements [47] of Trp-cage in the 1ϩ charge state, which infer the presence of a salt bridge structure and MD calculations [48] on the 1ϩ state that characterize the Trp-cage structure with a salt bridge present.…”

Section: Trp-cage Proteinmentioning

confidence: 86%

Fluorescence lifetime probe of biomolecular conformations

Shi¹,

Parks²

2010

Methods have been developed to measure the fluorescence lifetime versus temperature of trapped biomolecular ions derivatized with a fluorescent dye. Previous measurements for different sequences of polyproline peptides demonstrated that quenching rates are related to conformations and their spatial fluctuations. This paper presents the results of extending these methods to study the conformational dynamics of larger biomolecules. Vancomycin-peptide noncovalent complexes in the 1ϩ charge state were studied as a function of temperature for different W-KAA peptide chiralities (L-LDD, D-LDD, L-DLL). Fluorescence-quenching rates, k q , were found to be stereoselective for these different chiralities with relative magnitudesThe variation in fluorescent quenching resulting from switching the chirality of the single Trp residue was readily detectable. Molecular dynamics analysis of complexes formed by W-KAA (L-LDD) and W-KAA(L-DLL) indicates that increased flexibility in the (L-DLL) complex is correlated with reduced quenching rates. Fluorescence measurements were also performed for the Trp-cage protein comparing quenching rates in the 1ϩ, 2ϩ, and 3ϩ charge states for which k q. Measurements of a sequence including a single-point mutation infer the presence of a salt-bridge structure in the 1ϩ charge state and its absence in both the 2ϩ and 3ϩ states. Molecular dynamics structures of Trp-cage indicate that a salt bridge in the 1ϩ charge state produces more compact conformations leading to larger quenching rates based on the quenching mechanism. In both these experimental studies the fluorescence-quenching rates were consistent with changes in structure induced by either intermolecular or intramolecular interactions. (J Am Soc Mass Spectrom 2010, 21, 707-718) © 2010 American Society for Mass Spectrometry P revious measurements [1] of the lifetime of a fluorescent dye covalently bound to polypeptide ions exhibited a quenching rate that depends both on the separation of the dye to a Trp side chain and on the strength of electrostatic fields in the vicinity of the dye. Quenching rates were found to depend on specific peptide sequences giving rise to different conformations and spatial fluctuations characterized by each conformation. Consequently, lifetime measurements provide an opportunity to extract information sensitive to local molecular arrangements and, perhaps more importantly, changes in that arrangement determined by intramolecular and intermolecular interactions. This paper applies lifetime measurements to study two larger biomolecules: the vancomycin-peptide noncovalent complex and the Trp-cage protein. The structures of these biomolecules have been identified in NMR solution measurements; and each has been studied previously in gas-phase measurements. Results of gas-phase studies of the vancomycin-peptide complex infer that specific changes in the peptide ligand chirality lead to different hydrogen binding networks and thus to different conformations. Gas-phase measurements and calculations of Trp-cage protein i...