Quantum Cascade Laser Infrared Spectroscopy for Glycan Analysis of Glycoprotein Solutions

Glycans are oligosaccharides attached to proteins or lipids and affect their functions, such as drug efficacy, structural contribution, metabolism, immunogenicity, and molecular recognition. Conventional glycosylation analysis has relied on destructive, slow, system-sensitive methods, including enzymatic reactions, chromatography, fluorescence labeling, and mass spectrometry. Herein, we propose quantum cascade laser (QCL) infrared (IR) spectroscopy as a rapid, nondestructive method to quantify glycans and their monosaccharide composition. Previously, we demonstrated high-sensitivity IR spectroscopy of protein solution using solvent absorption compensation (SAC) and double-beam modulation (DBM) techniques. However, the SAC-DBM approach suffered a limited frequency scanning range (<400 cm^-1) due to the light dispersion by acousto-optic modulators (AOMs). Here, we implemented a mirror-based double-pass AOM in the SAC-DBM scheme and successfully extended the frequency range to (970 to 1840 cm^-1), which encompasses the vibrational fingerprint of biomolecules. The extended frequency range allowed the simultaneous observation of monosaccharide ring bands (1000 to 1200 cm^-1) and protein amide bands (1500 to 1700 cm^-1). We compared the IR spectra of six glycoproteins and two nonglycosylated proteins with the results from intact mass spectrometry. The IR absorbance ratios of the ring band to the amide band of glycoproteins in solutions showed a linear correlation with the ratios of glycan to protein backbone masses. Furthermore, a multivariate analysis produced monosaccharide compositions consistent with the reported database for the glycoproteins, and the monosaccharide compositions were used to improve the predictability of the glycan-protein mass ratio from the IR-absorbance ratio. This nondestructive, high-sensitivity QCL-IR spectroscopy could be used as a standard method to monitor batch-to-batch comparability during drug manufacturing and quantify the glycosylation and monosaccharide composition of new glycoproteins and other glycosylated biosystems.