Novel Approaches in Cold Ion Spectroscopy for Structural Characterization of Biomolecules in the Gas Phase

Mass spectrometry is a powerful analytical technique, which has become invaluable across a wide range of scientific fields, such as "omics" sciences (e.g., proteomics), doping control and forensic sciences, drug development, environmental analysis, geologic research, etc. Originally developed over a century ago to measure elemental atomic weights and the natural abundances of isotopes, nowadays mass spectrometry is a vital tool for structural characterization of biomolecules, ranging from small metabolites to large macromolecular assemblies. This includes identifying unknown compounds, quantifying known compounds, and exploring molecular structures. However, the mass spectrometric analysis is based on measurements of the mass-to-charge ratio of ions, a quantity that is not sensitive to their three dimensional (3D) structure. Cold ion spectroscopy is another method of studying ions in the gas phase, that has demonstrated its capability to reveal their spectroscopic fingerprints, which are characteristic of the 3D structure of the ions on a fundamental level. In this thesis we present novel approaches to molecular structure identification, which are based on integration of high-resolution mass spectrometry (MS) with high-resolution ultraviolet (UV) photofragmentation spectroscopy of cold ions. The first part of this work describes the coupling of an Exactive Orbitrap-based mass spectrometer to a cold ion spectrometer and the use of this novel hybrid instrument for measuring two-dimensional (2D) UV-MS spectra of ionic species. The synergy of the two complementary techniques makes these spectra unique fingerprints of biomolecular ions. The second part presents an approach that uses mathematical analysis of these highly specific fingerprints for identification of isomeric biomolecules in mixtures. Using preliminarily recorded libraries of fingerprints, the UV-MS approach was successfully employed for quantitative identification of positional isomers of a phosphorylated peptide, diastereomers of an opioid peptide, and diastereomers of a drug molecule. We also demonstrate a library-free approach, which allows estimating the number of components present in a mixture and deriving their UV absorption and fragmentation mass spectra directly from the 2D UV-MS spectrum of the mixture. In the third part, we employ the library-free approach and the phenomenon of nonstatistical photofragmentation to identify conformers of gas-phase biomolecular ions and to recover their individual UV absorption and fragmentation mass spectra. We validate this approach with a benchmark dipeptide, Tyr-Ala, and then apply it to a decapeptide, gramicidin S, and a small drug molecule, homatropine. A careful analysis of the derived UV and mass spectra of the conformers of gramicidin S and homatropine provides some structural information that can be used for solving their 3D structures. In the last part, we demonstrate a use of 2D UV-MS spectroscopy for estimating the distances between the phenylalanine and tyrosine chromophores in four gas-phase opioid peptides. We have also calculated the 3D structures of their lowest energy conformers. We discuss these structures in the context of the estimated interchromophore distances and the pharmacological properties of the peptides.

Cold ion spectroscopy for structural identifications of biomolecules

Oleg Boyarkine

Over the last decade, the spectroscopy of cryogenically cold ions iso- lated in the gas phase has been developed as a new tool for structural elucidations of biological molecules. Cooling allows for vibrational resolution in UV and IR spectra of small to midsize peptides, enabling different multi-laser techniques of conformer-specific spectroscopy. In conjunction with quantum chemistry calculations, IR spectra of sin- gle conformers allows for solving their intrinsic geometries. Here, we briefly review some fundamental and technical aspects of the cold- ion spectroscopy (CIS) approach and illustrate its application for pro- tonated peptides, carbohydrates and for non-covalent complexes of biomolecules. The challenges and limitations of CIS in view of its rele- vance to life-science studies are critically assessed. Finally, we discuss and illustrate some approaches of CIS for the analytical identification of biomolecules, in particular the recently developed method of 2D UV-MS fingerprinting, which combines CIS with high-resolution mass spectrometry.

2018

Cold Ion Spectroscopy of Biological Molecules in the Gas Phase

Natalia Nagornova Boyarkine

The three-dimensional (3D) structures of proteins and peptides in vivo largely determine their biological functions. In vitro these native structures and their heterogeneity reflect a subtle balance between noncovalent intramolecular interactions and those with the surrounding solvent molecules. Decoupling the intra- and intermolecular interactions and revealing the intrinsic structures of biomolecules in the gas phase is crucial for understanding protein-peptide (-protein, -membrane) binding processes and protein folding, and can assist in silico drug design. In addition to this, the spectroscopic studies of (bio)molecules in the gas phase provide crucial information that serves as a benchmark for validation of theoretical approaches, used for structural computations. In this thesis report we demonstrate the use of conformer-selective, Cold Ion Spectroscopy (CIS), combined with mass spectrometry and electrospray ionization technique, to reveal spectroscopic fingerprints of closed-shell (bio)molecular species in the gas phase. These fingerprints provide a stringent test for validating calculated structures of the molecules. Electronic and conformer-specific vibrational spectra of charged molecules, collisionally cooled in a linear radio frequency 22-pole ion trap have been measured using photofragment detection schemes that employs UV and/or IR laser light. A detailed set of spectroscopic and structural constraints, obtained using CIS method for an antibiotic peptide, gramicidin S (GS) allows an unambiguous determination of its 3D structure in the gas phase. The calculated gas-phase structure of GS differs substantially from its known structure in the condensed phase and, thus, provides complementary information for modeling biological activity of this antibiotic. At the next step we form hydrogen-bonded complexes of GS with a gradually increasing number of water molecules. Spectroscopy of such peptide-water clusters targets to bridge the gap between the gas phase and the in vitro structures. We have shown that both, UV and IR spectra of large GS-water clusters remain vibrationally resolved, if the species are cooled to cryogenic temperatures. The observed detailed spectroscopic fingerprints of the hydrated peptides provide a benchmark for their structural analysis. While CIS method was developed for biological species, here we demonstrate its successful application for structural determination of highly-reactive catalytic metalorganic species. The experimentally observed lack of a characteristic NH stretch vibration in the IR spectra of such a species allowed an unambiguous selection of its structures from several low-energy calculated candidates. The current approach for solving structures of biomolecules implies a comparison of a measured spectroscopic fingerprint with that, calculated for a candidate structure. Unfortunately, high accuracy structural computations become time consuming and challenging for large peptides. We revealed a specificity in electronic spectra of phosphorylated peptides, which coordinates with the docking position of a phosphogroup in peptides. This observation, if confirmed for a large variety of peptides, may allow a prediction of phosphorylation sites without a call for calculations. Regarding a relative simplicity of UV spectroscopy we see a potential of this approach as an orthogonal analytical method that assists mass spectrometry in some puzzling cases of peptide analysis. At the end of this work we tried to push the limits of CIS approach towards very large species and, for the first time, measured IR spectrum of a bare protein, cytochrome c, in the gas phase.

EPFL2011

Spectroscopic studies of cold, gas-phase biomolecular ions

Thomas Rizzo

While the marriage of mass spectrometry and laser spectroscopy is not new, developments over the last few years in this relationship have opened up new horizons for the spectroscopic study of biological molecules. The combination of electrospray ionization for producing large biological molecules in the gas phase together with cooled ion traps and multiple- resonance laser schemes are allowing spectroscopic investigation of individual conformations of peptides with more than a dozen amino acids. Highly resolved infrared spectra of single conformations of such species provide important benchmarks for testing the accuracy of theoretical calculations. This review presents a number of techniques employed in our laboratory and in others for measuring the spectroscopy of cold, gas-phase protonated peptides. We show examples that demonstrate the power of these techniques and evaluate their extension to still larger biological molecules.

2009

Cold Ion Spectroscopy of Biological Molecules in the Gas Phase

Natalia Nagornova Boyarkine

EPFL2011