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

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.

Combining Ion Mobility and Cryogenic Spectroscopy for Structural and Analytical Studies of Biomolecular Ions

Michael Zachary Kamrath, Thomas Rizzo

Ion mobility spectrometry (IMS) has become a valuable tool in biophysical and bioanalytical chemistry because of its ability to separate and characterize the structure of gas-phase biomolecular ions on the basis of their collisional cross section (CCS). Its importance has grown with the realization that in many cases, biomolecular ions retain important structural characteristics when produced in the gas phase by electrospray ionization (ESI). While a CCS can help distinguish between structures of radically different types, one cannot expect a single number to differentiate similar conformations of a complex molecule. Molecular spectroscopy has also played an increasingly important role for structural characterization of biomolecular ions. Spectroscopic measurements, particularly when performed at cryogenic temperatures, can be extremely sensitive to small changes in a molecule’s conformation and provide tight constraints for calculations of biomolecular structures. However, spectra of complex molecules can be heavily congested due to the presence of multiple stable conformations, each of which can have a distinct spectrum. This congestion can inhibit spectral analysis and complicate the extraction of structural information. Even when a single conformation is present, the conformational search process needed to match a measured spectrum with a computed structure can be overwhelming for peptides of more than a few amino acids, for example. We have recently combined ion mobility spectrometry and cryogenic ion spectroscopy (CIS) to characterize the structures of gas-phase biomolecular ions. In this Account, we illustrate how the coupling of IMS and CIS is by nature synergistic. On the one hand, IMS can be used as a conformational filter to reduce spectral congestion that arises from heterogeneous samples, facilitating structural analysis. On the other hand, highly resolved, cryogenic spectra can serve as a selective detector for IMS that can increase the effective resolution and hence the maximum number of distinct species that can be detected. Taken together, spectra and CCS measurements on the same system facilitates structural analysis and strengthens the conclusions that can be drawn from each type of data. After describing different approaches to combining these two techniques in such a way as to simplify the data obtained from each one separately, we present two examples that illustrate the type of insight gained from using spectra and CCS data together for characterizing gas-phase biomolecular ions. In one example, the CCS is used as a constraint for quantum chemical structure calculations of kinetically trapped species, where a lowest-energy criterion is not applicable. In a second example, we use both the CCS and a cryogenic infrared spectrum as a means to distinguish isomeric glycans.

2018

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

Gas-phase probes of kinetically trapped peptides

Liudmila Voronina

Gas-phase techniques are promising for studying intrinsically disordered peptides and proteins, as they can be implemented in a conformer-selective way. One of the issues that prevent them from finding a broader use is the question of to which extent the structure of a biological molecule is preserved when transferring it from solution to the gas phase via electrospray. We focus on cis/trans isomerization of prolyl-peptide bonds, which is known to have high barrier on the potential energy surface. Upon electrospraying, solution-like structures can find themselves kinetically trapped behind these barriers, as we explicitly show for the case of bradykinin 1-5 fragment. Using nuclear magnetic resonance, we determine that it adopts mostly all-trans conformation in solution, while three distinct conformational families are observed in the ion mobility experiment in the gas phase. Cryogenic ion spectroscopy combined with first-principles simulations allows us to identify the major conformers and show that the solution structural preferences of the prolyl-peptide bonds are preserved in the kinetically trapped conformational family, while the lowest-energy gas-phase structures have one of the prolines in the cis conformation. We show how collisional cross-sections and infrared spectra are used to guide and verify the calculations, giving rise to a new type of symbiosis between theory and experiment. This is especially valuable for the kinetically trapped structures, as in this case the energy criterion cannot be decisive. As a next step, we characterize the full nonapeptide bradykinin in the +3 charge state. Using field-asymmetric ion mobility spectrometry combined with cryogenic ion spectroscopy, we demonstrate the presence of three major conformational families in the gas phase, one of which is kinetically trapped. This result is in agreement with the drift-tube ion mobility data published previously, and we propose a correspondence between the conformational families separated by field-asymmetric and drift-tube ion mobility spectrometry. We obtain conformer-specific infrared spectra of the major conformers and assign the vibrational bands using 15N isotopic labeling. Substituting carbon atoms in the phenyl ring with their 13C isotope allows us to separate two types of structures according to their fragmentation pattern upon electronic excitation. This method can serve as a complementary way to introduce conformer-selectivity when studying large molecules. Finally, we assess the result of substituting one or two prolines in the bradykinin sequence with alanine. This method has been used previously to assign the peaks in the drift-time distributions to certain conformations of the prolyl-peptide bonds. We show that the mutants do reproduce a part of the conformers of the original peptide, but also form additional structures due to the higher flexibility of the alanine backbone compared to proline. We conclude that the proline-to-non-proline substitutions are helpful to assign the structures, but have to be used in conjunction with spectroscopic techniques, which allow detailed comparison of the structures of the mutant and the native peptide. In general, this work is one of the first steps towards a database of atomic-resolution peptide structures in the gas phase and a better understanding of the capabilities and limits of spectroscopy and ion mobility in the gas phase applied to intrinsically disordered peptides.

EPFL2016