Combining Cryogenic Infrared Spectroscopy with Selective Enzymatic Cleavage for Determining Glycan Primary Structure

Given the biological relevance and intrinsic structural complexity of glycans, increasing efforts are being directed towards developing a general glycan database that includes information from different analytical methods. As recently demonstrated, cryogenic infrared (IR) spectroscopy is a promising technique for glycan analysis, as it provides unique vibrational finger-prints of specific glycan isomer ions. One of the main goals of a glycan database is the identification and detailed characteri-zation of unknown species. In this work, we combine enzymatic digestion with cryogenic IR-spectroscopy and demonstrate how it can be used for glycan identification. We measured the IR-spectra of a series of cationic glycan standards of increasing complexity and compared them with spectra of the same species after enzymatic cleavage of larger glycans. We show that the cryogenic IR spectra of the cleaved glycans are highly structured and virtually identical to those of standards after both single and multiple cleavages. Our results suggest that the combination of these methods represents a potentially powerful and spe-cific approach for the characterization of unknown glycans