Beyond Fourier Transform Infrared Spectroscopy: External Cavity Quantum Cascade Laser-Based Mid-infrared Transmission Spectroscopy of Proteins in the Amide I and Amide II Region

Abstract

In this work, we present a setup for mid-IR measurements of the protein amide I and amide II bands in aqueous solution. Employing a latest generation external cavity-quantum cascade laser (EC-QCL) at room temperature in pulsed operation mode allowed implementing a high optical path length of 31 μm that ensures robust sample handling. By application of a data processing routine, which removes occasionally deviating EC-QCL scans, the noise level could be lowered by a factor of 4. The thereby accomplished signal-to-noise ratio is better by a factor of approximately 2 compared to research-grade Fourier transform infrared (FT-IR) spectrometers at equal acquisition times. Employing this setup, characteristic spectral features of three representative proteins with different secondary structures could be measured at concentrations as low as 1 mg mL. Mathematical evaluation of the spectral overlap confirms excellent agreement of the quantum cascade laser infrared spectroscropy (QCL-IR) transmission measurements with protein spectra acquired by FT-IR spectroscopy. The presented setup combines performance surpassing FT-IR spectroscopy with large applicable optical paths and coverage of the relevant spectral range for protein analysis. This holds high potential for future EC-QCL-based protein studies, including the investigation of dynamic secondary structure changes and chemometrics-based protein quantification in complex matrices.

Custom made inclusion bodies: impact of classical process parameters and physiological parameters on inclusion body quality attributes

“…Infrared (IR) spectra were recorded by an external-cavity quantum cascade laser-based IR transmission setup described in detail by Schwaighofer et al [ 31 ]. A water-cooled external-cavity quantum cascade laser (Hedgehog, Daylight Solutions Inc., San Diego, USA) was used operating at a repetition rate of 100 kHz and a pulse width of 5000 ns.…”

“…Quality attributes (or key performance indicator) of IBs, such as titer and morphology changes during extraction procedures have already been studied and show that IBs are dynamic structures depending on the cultivation and extraction conditions [ 27 – 29 ]. First approaches towards IB sizing in the upstream process have already been made within our group by Reichelt et al [ 30 ] using transmission electron microscopy [ 31 ] in combination with nanoparticle tracking analysis (NTA) revealing general trends of IB growth during cultivation. Further studies show that IBs consist of up to 50% correctly folded protein in contrast to the general perception of IBs as inactive structures [ 29 , 32 ].…”

Sensitive Spectroscopy of Acetone Using a Widely Tunable External-Cavity Quantum Cascade Laser

“…Standard Fourier transform infrared spectroscopy (FTIR) is considered as a benchmark due to its broadband spectral coverage spanning over the entire mid-infrared spectral region and highly accurate wavelength scale. In addition, the spectra can be averaged for a long time to reach high detection sensitivities [ 18 ]. However, FTIR systems are bulky, expensive, have a short interaction path length and require a long acquisition/averaging time to achieve high sensitivity.…”

“…Specifically, rotation of the grating in the external cavity introduces mode-hops due to the change of the laser cavity length and results in a spectral mismatch in the recordings of consecutive scans. Consequently, in the broadband tuning of the EC-QCL, low noise levels are hard to achieve due to the difficulty of averaging the consecutive scans efficiently [ 18 ]. This effect is reduced significantly in the narrowband scan which is performed by tuning the piezo actuator(s) mounted on the diffraction grating.…”

A New Quantum Cascade IR‐Laser Online Detector: Chemical‐Sensitive Size‐Exclusion Chromatography Measurement at Unprecedented Low Levels

“…As this is an online coupled technique, the time for the IR measurements is also limited (e.g., 15 s) to retain the resolution in the SEC size separation. Comparable method development work was done by Lendl and co‐workers where they coupled liquid adsoption chromatography, LAC, to a QCL setup to measure sugars in beverages and later stand‐alone QCL setups were used to measure biological applications such as proteins in aqueous solutions, glucose levels, and multianalyte blood analysis . Analog correlation between chemical composition and molecular weight are currently conducted via SEC‐NMR indicating the needs for the acquisition of correlated information .…”