Cryogenic Vibrational Spectroscopy of Conformationally Selected Biomolecules
Vibrational spectroscopy of biological molecules is complicated by the coexistence of multiple stable conformers and/or isomers, which causes spectral congestion and hinders analysis, even at low temperatures. Spectroscopic schemes such as IR-UV double resonance or IR-IR hole burning can help deal with this conformational or isomeric heterogeneity in favorable cases, but neither is universally applicable. New spectral simplification methods are thus needed to push vibrational spectroscopy to increasingly complex biological molecules in a meaningful way. One class of molecules for which this is particularly important is that of glycans, or sugars.
Despite their biological importance, glycans present a particular problem for analysis arising from the multiple types of isomerism that are simultaneously present. Orthogonal techniques need to be applied simultaneously to have any chance of resolving their isomeric complexity. The most common approach is to combine liquid chromatography (LC) with mass spectrometry (MS), but LCMS cannot distinguish the subtlest isomeric forms. Ion mobility spectrometry (IMS) is being increasingly applied to glycan analysis, but even this powerful technique cannot completely distinguish subtly different isomers. Vibrational spectroscopy, on the other hand, is exquisitely sensitive to the smallest difference between isomeric species, particularly when performed at cryogenic temperatures.
We have recently developed a technique that combines ultrahigh-resolution ion mobility spectrometry with cryogenic ion vibrational spectroscopy for the analysis of gas-phase biological ions, and we have been applying it principally to glycans (1-3). We achieve extremely high-resolution ion mobility using structures for lossless ion manipulations (SLIM) (4), which employs traveling-waves generated between a closely spaced pair of printed circuit-board electrodes. Because ions can be made to follow a serpentine path, one can achieve ultra-long separation lengths, and hence extremely high resolution, in a compact instrument. This allows us to separate glycan isomers that differ only by the orientation about a single stereogenic center. After mobility separation, we then record a vibrational fingerprint at cryogenic temperatures using messenger-tagging spectroscopy and compare it with a database constructed from suitable standards.
We will demonstrate here the power of this technique by applying it to a series of glycans of increasing complexity and show the steps we are taking towards making this approach into a high-throughput analytical tool.
(1) Warnke, S.; Ben Faleh, A.; Scutelnic, V.; Rizzo, T. R. J. Am. Soc. Mass Spectrom. 2019, 30, 2204-2211.
(2) Warnke, S.; Ben Faleh, A.; Pellegrinelli, R. P.; Yalovenko, N.; Rizzo, T. R. Faraday Discuss. Chem. Soc. 2019.
(3) Ben Faleh, A.; Warnke, S.; Rizzo, T. R. Anal. Chem. 2019, 91, 4876-4882.
(4) Hamid, A. M.; Garimella, S. V. B.; Ibrahim, Y. M.; Deng, L.; Zheng, X.; Webb, I. K.; Anderson, G. A.; Prost, S. A.; Norheim, R. V.; Tolmachev, A. V.; Baker, E. S.; Smith, R. D. Anal. Chem. 2016, 88, 8949-8956.