Spectroscopy of mobility-selected biomolecular ions

We describe here experiments that combine differential ion mobility, which separates conformational isomers of biomolecular ions, with electronic spectroscopy in a cold, radio-frequency ion trap. Although the low temperature attainable in a cold ion trap greatly simplifies the electronic spectra of large molecules, conformational heterogeneity can still be a significant source of congestion, complicating spectroscopic analysis. We demonstrate here that using differential ion mobility to separate gas-phase peptide conformers before injecting them into a cold ion trap allows one to decompose a dense spectrum into contributions from different conformational families. In the inverse sense, cold ion spectroscopy can be used as a conformation-specific detector for ion mobility, allowing one to separate an unresolved peak into contributions from different conformational families. The doubly protonated peptide bradykinin serves as a good test case for the marriage of these two techiques as it exhibits a considerable degree of conformational heterogeneity that results in a highly congested electronic spectrum. Our results demonstrate the feasiblity and advantages of directly coupling ion mobility with spectroscopy and provide a diagnostic of conformational isomerization of this peptide after being produced in the gas phase by electrospray.

A Combination of Cold Ion Spectroscopy and Ion Mobility for the Study of Complex Peptides and Small Proteins

Georgios Papadopoulos

We present in this work experiments that push the limits of cold ion spectroscopy to the study of complex peptides and small proteins in the gas phase. Although the low temperature attainable in a cold ion trap greatly simplifies the electronic spectra of large molecules, conformational heterogeneity can still be a significant source of congestion, complicating spectroscopic analysis. To overcome this hurdle, we propose an in tandem combination of ion mobility spectrometry and photofragment spectroscopy. This permits for conformational separation prior to laser interrogation. Towards this end, we describe a proof-of-principle experiment where we combine high- Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) with electronic spectroscopy. We demonstrate that using FAIMS to separate gas-phase peptide conformers before injecting them into a cold ion trap allows one to decompose a dense spectrum into contributions from different conformational families. In the inverse sense, cold ion spectroscopy can be used as a conformation-specific detector for ion mobility, allowing one to separate an unresolved mobility peak into contributions from different conformational families. The doubly protonated peptide bradykinin serves as a good test case for the marriage of these two techniques as it exhibits a considerable degree of conformational heterogeneity that results in a highly congested electronic spectrum. Our results demonstrate the feasibility and advantages of directly coupling ion mobility with spectroscopy and provide a diagnostic of conformational isomerization of this peptide after being produced in the gas phase by electrospray. In a second series of studies we show the potential but also the limitations of our spectroscopic approach for investigating small, naturally occurring proteins, by applying it to ubiquitin, a protein of 76 amino acids. We present electronic photofragment spectra of various protonation states of ubiquitin and we compare our results to literature data acquired by ion mobility spectrometry. These experiments show that the information obtained by laser interrogation of molecules of this size is limited and could be enhanced by a combination with ion mobility techniques. Although some of the results obtained may be important in characterizing small proteins in the gas phase, these experiments are at an early stage and the application of photofragment spectroscopy on proteins needs more investigation.

EPFL2012

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

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.