Cryogenic Ion Spectroscopy for Identification of Monosaccharide Anomers

We combine conformer-selective, cryogenic infrared spectroscopy, quantum mechanical computations, and 18O substitution at the reducing end to determine the structural preferences of protonated glucosamine in the gas phase. Cryogenic infrared-infrared (IR-IR) double resonance spectroscopy of helium-tagged protonated glucosamine provides vibrational fingerprints of individual conformers, and 18O isotopic labeling facilitates the match with computed structures and provides a selective probe of the anomeric hydroxyl. This is key for using vibrational spectroscopy for glycan analysis and determining the generality of anomeric memory during glycosidic bond cleavage.

Identification of Mobility-Resolved N-Glycan Isomers

Priyanka Bansal, Ahmed Ben Faleh, Robert Paul Pellegrinelli, Thomas Rizzo, Stephan Warnke

ABSTRACT: Glycan analysis has evolved considerably during the last decade. The advent of high-resolution ion-mobility spectrometry has enabled the separation of isomers with only the slightest of structural differences. However, the ability to separate such species raises the problem of identifying all the mobility-resolved peaks that are observed, especially when analytical standards are not available. In this work, we report an approach based on the combination of IMSn with cryogenic vibrational spectroscopy to identify N-glycan reducing-end anomers. By identifying the reducing-end alpha and beta anomers of diacetyl-chitobiose, which is a disaccharide that forms part of the common core of all N-glycans, we are able to assign mobility peaks to reducing anomers of a selection of N-glycans of different sizes, starting from trisaccharides such as Man-1 up to glycans containing nine monosaccharide units, such as G2. By building an infrared fingerprint database of the identified N-glycans, our approach allows unambiguous identification of mobility peaks corresponding to reducing-end anomers and distinguishes them from positional isomers that might be present in a complex mixture.

AMER CHEMICAL SOC2022

A New Strategy Coupling Ion-Mobility-Selective CID and Cryogenic IR Spectroscopy to Identify Glycan Anomers

Priyanka Bansal, Ahmed Ben Faleh, Eduardo Carrascosa Casado, Robert Paul Pellegrinelli, Thomas Rizzo, Stephan Warnke, Lei Yue

Determining the primary structure of glycans remains challenging due to their isomeric complexity. While high-resolution ion mobility spectrometry (IMS) has recently allowed distinguishing between many glycan isomers, the arrival-time distributions (ATDs) frequently exhibit multiple peaks, which can arise from positional isomers, reducing-end anomers, or different conformations. Here, we present the combination of ultrahigh-resolution ion mobility, collision-induced dissociation (CID), and cryogenic infrared (IR) spectroscopy as a systematic method to identify reducing-end anomers of glycans. Previous studies have suggested that high-resolution ion mobility of sodiated glycans is able to separate the two reducing-end anomers. In this case, Y-fragments generated from mobility-separated precursor species should also contain a single anomer at their reducing end. We confirm that this is the case by comparing the IR spectra of selected Y-fragments to those of anomerically pure mono- and disaccharides, allowing the assignment of the mobility-separated precursor and its IR spectrum to a single reducing-end anomer. The anomerically pure precursor glycans can henceforth be rapidly identified on the basis of their IR spectrum alone, allowing them to be distinguished from other isomeric forms.

2022

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

Gas-phase probes of kinetically trapped peptides

Liudmila Voronina

EPFL2016