Toward High-Throughput Cryogenic IR Fingerprinting of Mobility-Separated Glycan Isomers

[Image: see text] Infrared (IR) spectroscopy is a powerful tool used to infer detailed structural information on molecules, often in conjunction with quantum-chemical calculations. When applied to cryogenically cooled ions, IR spectra provide unique fingerprints that can be used for biomolecular identification. This is particularly important in the analysis of isomeric biopolymers, which are difficult to distinguish using mass spectrometry. However, IR spectroscopy typically requires laser systems that need substantial user attention and measurement times of tens of minutes, which limits its analytical utility. We report here the development of a new high-throughput instrument that combines ultrahigh-resolution ion-mobility spectrometry with cryogenic IR spectroscopy and mass spectrometry, and we apply it to the analysis of isomeric glycans. The ion mobility step, which is based on structures for lossless ion manipulations (SLIM), separates glycan isomers, and an IR fingerprint spectrum identifies them. An innovative cryogenic ion trap allows multiplexing the acquisition of analyte IR fingerprints following mobility separation, and using a turn-key IR laser, we can obtain spectra and identify isomeric species in less than a minute. This work demonstrates the potential of IR fingerprinting methods to impact the analysis of isomeric biomolecules and more specifically glycans.