Spectroscopic studies of peptide fragments produced by collision-induced dissociation

Tandem mass spectrometry (MS/MS) combined with collision-induced dissociation (CID) is a key method for primary structure determination of peptides and proteins. Collisionally activated peptides undergo statistical dissociation, forming a series of backbone bn and yn fragment ions that reflect the peptide sequence. However, nascent CID fragments may undergo sequence permutation via "head-to-tail" cyclization, and upon further dissociation give rise to non-direct fragments, hindering the mass spectral interpretation. In this thesis work laser spectroscopy in a cold ion trap is used to provide structural information on the nascent fragments, which can help elucidate the fragmentation mechanisms and predict the occurrence of sequence permutation. The first part of the work is devoted to the b6 fragment ion formed from the protonated model peptide [FAGFAGPG + H]+. For this species five unique conformations have been identified under our experimental conditions. Nitrogen-15 isotopic substitution of individual amino acid residues reveals two intriguing totally symmetric cyclic species. This analysis also provides unambiguous evidence for proton migration during CID for the remaining asymmetric conformers. This information, as well as the vibrational band assignments, simplified the conformational search performed by simulated annealing combined with DFT calculations. The superb agreement between experiment and theory provided a high degree of confidence in the determined structures. To our knowledge this is the first conformer-selective spectroscopic identification of the cyclic bn ions implicated in sequence scrambling during CID. The second part describes the experiments in which the barriers to conformational isomerization have been probed by IR hole-filling spectroscopy (HFS). The HFS recorded in the fundamental as well as in the first overtone NH stretch vibration regions are presented. The next part describes the experiments in which the influence of amino acid composition and length of bn ions on their structural aspects have been investigated. The b6 ions from [FAVFAGPG + H]+ and [YAGYAGPG + H]+ are described first. Based on the comparison of their IR-UV spectra with those of b6 [FAGFAGPG + H]+, some structural features of the former are deduced. For the larger b8 ion from [FAVGFAVGPG + H]+, we have identified six unique conformers, one of which has a totally symmetric cyclic structure. The last part describes construction and characterization of a new cryogenic octopole ion trap. Its advantages over the previously used 22-pole trap are demonstrated.

Electronic and vibrational spectroscopy of cold protonated amino acids in the gas phase

Sébastien Mercier

In this thesis report, we describe a novel home-built instrument designed to study the spectroscopy of biomolecular ions in the gas phase and at low temperature, and we present the first experimental results obtained on protonated aromatic amino acids. The apparatus consists in a tandem mass spectrometer equipped with a nanospray ion source and a cryocooled 22-pole ion trap. The charged species produced by the source traverse a first quadrupole mass filter and mass-selected ions are then accumulated and thermalized in the ion trap, where their vibrational temperature can be lowered to ~ 10 K. Two setups allow us to generate ultraviolet and infrared laser light to spectroscopically probe the trapped ions by photodissociation. The resulting fragment ions are finally released from the trap and mass analyzed by a second quadrupole spectrometer. Photodissociation electronic spectra have been measured for the protonated amino acids tryptophan (TrpH+) and tyrosine (TyrH+), as well as for hydrated complexes of the former. The spectrum of TyrH+ exhibits sharp, fully resolved vibronic transitions, which attests to the low temperature of the ions. In contrast, that of tryptophan shows a broad absorption band that covers several hundreds of wavenumbers. This has been attributed to lifetime broadening related to an ultrafast (< ~ 100 fs) deactivation mechanism of the TrpH+ S1(ππ*) excited state. This phenomenon is caused by a strong coupling between this ππ* state and a nearby dissociative state of πσ* character mainly localized on the charged ammonium group. Solvation of TrpH+ by only two water molecules is sufficient to significantly destabilize the σ* orbitals and reduce that coupling, which results in a longer excited-state lifetime and a fully resolved electronic spectrum. These interpretations are supported by ab initio and density functional theory (DFT) calculations. Infrared-ultraviolet double resonance spectroscopic techniques have been successfully implemented to identify the different conformers of TyrH+ and doubly hydrated protonated tryptophan (TrpH+·W2) by measuring conformer-specific vibrational and electronic spectra for each of them. By comparison with the results of DFT geometry and harmonic frequency calculations, we could confidently determine the structures of the TyrH+ conformers and suggest possible assignments for those of TrpH+·W2. Two important stabilizing interactions could be inferred from these results: (i) the attraction between the charged ammonium group and the π-electron cloud of the aromatic ring; (ii) the hydrogen-bonding interaction of a donor water molecule to the acceptor indole ring of TrpH+·W2.

EPFL2008

Cryogenic Ion Spectroscopy of Peptides and Glycans

Chiara Masellis

Understanding the structure of biomolecules, which are directly related to physiological processes that take place in the human body, has always generated a large interest in the scientific community. In this thesis we focus our attention on two of these classes of biomolecules: peptides and glycans. In the first part of this work we investigate how the structure of helical peptides in the gas-phase is modified when one increases the number of amino acids in the sequence. Moreover, we analyze the role played by a mobile charge in the formation of the helical motif in the gas-phase. To achieve these goals, we make use of a home-built tandem mass spectrometer, and a pump-probe laser spectroscopic scheme. The combination of infrared and ultraviolet light together with cryogenic temperatures allows one to collect high-resolution conformer-specific spectra of gas-phase peptides. By comparing the experimental results obtained with density functional theory calculations, we show that the synergy between theory and experiment is key to provide accurate structural characterization of the investigated peptides. The mobile charge, in fact, induces unconventional backbone conformations, which cannot be predicted a priori by performing experiments alone. In the second part of this thesis we present a novel technique to identify and characterize glycans in the gas-phase that exploits a database approach. Due to their structural heterogeneity, glycans pose a problem for the currently available mass- and mobility-spectrometry techniques. In the work herein we show that we can easily identify glycan structural isomers, by adding a spectroscopic dimension to mass and mobility measurements. Cryogenic vibrational messenger spectroscopy, in fact, allows us to unambiguously identify disaccharides and pentasaccharides isomers due to the high-resolution provided by this method. Moreover, we can perform our experiments in a broad range of temperatures, going from liquid helium to liquid nitrogen. This shows the potential of our technique in becoming a more accessible analytical tool for glycan identification.

EPFL2017

A gas phase approach for glycan analysis: Combining ultrahigh-resolution ion mobility spectrometry with cryogenic vibrational spectroscopy

Ahmed Ben Faleh

The last decades have been punctuated by the development of revolutionary analytical technologies that have enabled the analysis of biomolecular structures and unraveled the roles they play in biological systems. However, certain classes of biomolecules remain less understood than others. In fact, despite being among the most abundant molecules on our planet, glycans remain among the least understood class of biological macromolecules due to their highly complex structure. The complexity of glycan molecules arises from their isomeric nature, which poses a significant challenge to classical analytical tools. The advent of ionization methods such as ESI and MALDI has led to an unprecedented expansion of gas phase techniques for the analysis of biomolecules. Compared to condensed phase approaches, gas phase analysis allows for isolating specific molecules and collecting highly accurate and specific structural information. In this work, we present an analytical approach for the analysis of glycan molecules based on the combination of different gas phase techniques. A central part of this thesis is the development, design, and characterization of two new instruments that have the capabilities to identify glycan isomers unambiguously. Our experimental approach is based on the combination of high-resolution ion mobility spectrometry with cryogenic IR spectroscopy. Ion mobility spectrometry allows to separate charged molecules in the gas phase according to their overall shape, serving as an isomer-selective prefilter to the IR spectroscopic interrogation process. While IMS provides a way to separate glycan isomers, vibrational spectroscopy is used to identify the mobility-separated molecules. The IR absorptions characteristic of glycan molecules lead to distinct spectroscopic fingerprints, which are perfectly suited for the identification of glycan isomers. In the first part of this work, we report the modification of an existing instrument that combined drift tube IMS with IR spectroscopy. We replaced the drift tube section by IMS device employing a relatively novel technique called SLIM, which provides the highest mobility resolution reported to date while ensuring a minimum loss of ions. This prototype instrument was used in a series of proof-of-concept experiments where we successfully demonstrate that our approach provides highly accurate results, comparable to sophisticated double-resonance spectroscopic schemes. We also describe an analytical protocol that serves to identify the isomeric content of disaccharide and tetrasacharide mixtures unambiguously, using a database approach. Furthermore, we report a series of experiments in which we separate and identify the anomers of disaccharides. In the second part of the thesis, we describe a second-generation instrument based on the same approach. The design of this instrument was aimed at maximizing its sensitivity and throughput while adding more analytical functionalities. It includes a CID section, as well as a cryogenic multi-trap allowing for the multiplexed spectroscopic analysis of glycan isomers. We hereby report the characterization results highlighting the instrument capabilities in terms of ion transmission and IMS resolution. We also report the first rapid, multiplexed, IR spectral acquisition of two tetrasacharide isomers, which emphasizes the unique capabilities of this home-built instrument.

EPFL2021