Vladimir Kopysov

Cryogenically cooled octupole ion trap for spectroscopy of biomolecular ions

We present here the design of a linear octupole ion trap, suitable for collisional cryogenic cooling and spectroscopy of large ions. The performance of this trap has been assessed using ultraviolet (UV) photofragmentation spectroscopy of protonated dipeptides. At the trap temperature of 6.1 K, the vibrational temperature of the ions reaches 9.1 K, although their estimated translational temperature is ~150 K. This observation suggests that, despite the significant translational heating by radio-frequency electrical field, vibrational cooling of heavy ions in the octupole is at least as efficient as in the 22-pole ion traps previously used in our laboratory. In contrast to the 22-pole traps, excellent radial confinement of ions in the octupole makes it convenient for laser spectroscopy and boosts the dissociation yield of the stored ions to 30%. Overlap of the entire ion cloud by the laser beam in the octupole also allows for efficient UV depletion spectroscopy of ion-He clusters. The measured electronic spectra of the dipeptides and the clusters differ drastically, complicating a use of UV tagging spectroscopy for structural determination of large species.

Conformational Structures of a Decapeptide Validated by First Principles Calculations and Cold Ion Spectroscopy

Calculated structures of the two most stable conformers of a protonated decapeptide gramicidin S in the gas phase have been validated by comparing the vibrational spectra, calculated from first- principles and measured in a wide spectral range using infrared (IR)-UV double resonance cold ion spectroscopy. All the 522 vibrational modes of each conformer were calculated quantum mechanically and compared with the experiment without any recourse to an empirical scaling. The study demonstrates that first-principles calculations, when accounting for vibrational anharmonicity, can reproduce high-resolution experimental spectra well enough for validating structures of molecules as large as of 200 atoms. The validated accurate structures of the peptide may serve as templates for in silico drug design and absolute calibration of ion mobility measurements.

Resonance Energy Transfer Relates the Gas-Phase Structure and Pharmacological Activity of Opioid Peptides

Enkephalins are efficient pain-relief drugs that bind to transmembrane opioid receptors. One key structural parameter that governs the pharmacological activity of these opioid peptides and is typically determined from condensed-phase structures is the distance between the aromatic rings of their Tyr and Phe residues. We use resonance energy transfer, detected by a combination of cold ion spectroscopy and mass spectrometry, to estimate the Tyr-Phe spacing for enkephalins in the gas phase. In contrast to the condensed-phase structures, these distances appear to differ substantially in enkephalins with different pharmacological efficiencies, suggesting that gas-phase structures might be a better pharmacophoric metric for ligand peptides.

Colors for Molecular Masses: Fusion of Spectroscopy and Mass Spectrometry for Identification of Biomolecules

We present an approach that integrates ultraviolet (UV) photofragmentation spectroscopy of cold ions with high-resolution Orbitrap mass spectrometry (MS) and uses mathematical analysis of the recorded 2D data arrays for structural identification of biomolecules. The synergy of the two orthogonal techniques makes these arrays unique fingerprints of molecular ions, enabling their reliable identifications. Using preliminary created libraries of fingerprints, the UV-MS approach was successfully applied for quantitative identification of exact isobaric molecules in their mixtures, which is one of the challenging cases for mass spectrometry. We also demonstrate how the UV and fragmentation mass spectra of unknown chemical components of a mixture can be recovered from its fingerprint even without a use of library.

Identification of tyrosine-phosphorylated peptides using cold ion spectroscopy.

The accurate and unambiguous detection of post-translational modifications in proteins and peptides remains a challenging task. We report here the use of cold ion spectroscopy for the identification of phosphorylated tyrosine residues in peptides. This approach employs the wavelength-specific UV fragmentation of cryogenically cooled protonated peptides in the gas phase. In addition to the appearance of specific photofragments, the phosphorylation of tyrosine induces large spectral shifts of the peptide electronic band origins. Quantum chemical calculations and experiments together suggest a certain generality of the use of such shifts in the spectroscopic identification of phosphotyrosines. The enhanced selectivity offered by the joint application of wavelength-specific fragmentation and mass spectrometry of cold molecules can also be used in the identifications of aromatic residues in protonated peptides and, potentially, of other UV-absorbing groups in a variety of large polyatomic ions.

Nonstatistical UV Fragmentation of Gas-Phase Peptides Reveals Conformers and Their Structural Features.

Solving the 3D structure of a biomolecule requires recognition of its conformers and measurements of their individual structural identities, which can be compared with calculations. We employ the phenomenon of nonstatistical photofragmentation, detected by a combination of UV cold ion spectroscopy and high-resolution mass spectrometry, to identify the main conformers of gas-phase peptides and to recover individual UV absorption and mass spectra of all of these conformers in a single laser scan. We first validate this approach with a benchmark dipeptide, Tyr-Ala, and then apply it to a decapeptide, gramicidin S. The revealed characteristic structural difference between the conformers of the latter identifies some of the previously calculated structures of gramicidin S as the most likely geometries of its remaining unsolved conformer.

Initial steps of amyloidogenic peptide assembly revealed by cold ion spectroscopy

The early stages of fibril formation are difficult to capture in solution. We use cold-ion spectroscopy to examine an 11-residue peptide derived from the protein transthyretin and clusters of this fibre-forming peptide containing up to five units in the gas phase. For each oligomer, the UV spectra exhibit distinct changes in the electronic environment of aromatic residues in this peptide compared to that of the monomer and in the bulk solution. The UV spectra of the tetra- and pentamer are superimposable but differ significantly from the spectra of the monomer and trimer. Such a spectral evolution suggests that a common structural motif is formed as early as the tetramer. The presence of this stable motif is further supported by the low conformational heterogeneity of the tetra- and pentamer, revealed from their IR spectra. From comparison of the IR-spectra in the gas and condensed phases, we propose putative assignments for the dominant motif in the oligomers.

Identification of Isomeric Ephedrines by Cold Ion UV Spectroscopy: Toward Practical Implementation

Ephedrine and pseudoephedrine are stimulant drugs whose use is prohibited in athletic competition by the World Anti-Doping Agency (WADA) at very different threshold doping violation concentrations. We use a recently developed universal approach that integrates UV photofragmentation spectroscopy of cold ions with Orbitrap mass spectrometry (MS) for highly selective and highly sensitive identification of these diastereomers. Both species can be selectively detected at a solution concentration of a few tens of ng/mL, which is almost 3 orders of magnitude lower than the threshold concentration required by WADA. Relative concentrations of the isomers in solutions have been determined with the standard deviation of 3.1%, when the ions were cooled in an ion trap maintained at T = 6 K. Considering practical implementation of the method, we evaluated its performance for a simplified instrumentation. At an affordable elevated temperature of ∼70 K and with a low-maintenance midbandwidth optical parametric oscillator, a few second measurement should yield nearly the same selectivity and only ten times lower sensitivity than with the current research grade instrument.

High Susceptibility of Histidine to Charge Solvation Revealed by Cold Ion Spectroscopy

Histidine remained the last aromatic amino acid for which the intrinsic spectroscopic properties and structures were obscure. We measured the UV and IR spectra of protonated histidine, isolated in the gas phase, using photofragmentation cold ion spectroscopy. Unexpectedly, the UV absorption appears strongly redshifted relative to that of the cation in aqueous solutions. In investigating this phenomenon, we solved the geometries of all abundant conformers using IR conformer-selective spectroscopy and ab initio quantum chemical calculations. In all of the structures, the proton resides on the imidazole ring. The measured UV spectra of protonated methylimidazole, histamine and histidine, together with calculations of the electronic spectra for the latter, suggest that, in comparison with other aromatic amino acids, such location of proton makes UV absorption of histidine highly sensitive to the local environment of its side chain.

Peptide Bond Ultraviolet Absorption Enables Vibrational Cold-Ion Spectroscopy of Non-aromatic Peptides

Peptide-bond VUV absorption is inherent to all proteins and peptides. Although widely exploited in top-down proteomics for photodissociation, this absorption has never been spectroscopically characterized in the gas phase. We have measured VUV/UV photofragmentation spectrum of a single peptide bond in a cryogenically cold protonated dipeptide. Although the spectrum appears to be very broadband and structureless, vibrational pre-excitation of this and even larger cold peptides significantly increases the UV dissociation yield for some of their photofragments. We use this effect to extend the technique of IR-UV photofragmentation vibrational spectroscopy, developed for aromatic peptides, to nonaromatic ones and demonstrate measurements of conformation-specific and nonspecific IR spectra for di- to hexa-peptides.