Laser-based mid-infrared spectroscopy enables in-line detection of protein secondary structure from preparative liquid chromatography

Abstract

External cavity-quantum cascade laser (EC-QCL) based mid-infrared (IR) spectroscopy is an emerging technology for analyzing proteins in aqueous solutions. Higher sensitivity and larger applicable optical path lengths compared to conventional Fourier-transform IR (FTIR) spectroscopy open a wide range of possible applications, including near realtime protein monitoring from complex downstream operations. In this work, an EC-QCL based mid-IR spectrometer was coupled to a preparative liquid chromatography (LC) system. The large optical path length (25 μm) and the broad tuning range of the laser (1350-1750 cm^-1 ) allowed robust spectra acquisition in the most important wavenumber range for protein secondary structure determination. A model system based on size exclusion chromatography (SEC) and three different proteins was employed to demonstrate the advantages of LCQCL-IR coupling. The recorded spectra showed distinct amide I and II bands across the chromatographic run. Mid-IR spectra, extracted from the three chromatographic peak maxima showed features typical for the secondary structures of the exhibited proteins with high comparability to off-line reference spectra. Band positions and maxima of mid-IR absorbances were compared to a conventional UV detector, revealing excellent agreement of peak shapes and maxima. This work demonstrates that laser-based mid-IR spectroscopy offers the significant advantage of providing almost realtime information about protein secondary structure, which typically has to be obtained by laborious and time-consuming offline analysis. Consequently, coupling of LC and laser-based mid-IR spectroscopy holds high potential for replacing conventional off-line methods for monitoring proteins in complex biotechnological processes.