Technische Universität Wien
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EC-QCL based IR Transmission Spectroscopy of Proteins

Motivation

Mid-infrared spectroscopy is a well-established technique for the analysis of polypeptides and proteins. The amide I band (1700-1600 cm-1) originating from C=O stretching coupled to in-phase bending vibration of N-H and the amide II band (1600-1500 cm-1) arising from N-H bending and C-N stretching vibrations are the most useful bands for secondary structure evaluation and quantification of proteins.
Strong absorption of H2O near 1640 cm-1 makes IR studies of proteins in aqueous solutions challenging. For conventional Fourier-transform infrared spectroscopy, very short pathlength of approx. 10 µm are needed to avoid total IR absorption in the spectral region of water. These low path lengths limit the intensities of the IR bands and the signal-to-noise ratio, thus restricting the application to high protein concentrations (approx. 10 mg/mL).
EC-QCLs, which provide spectral power densities several orders of magnitude higher than thermal emitters,  allow the use of 4-5 times higher path length and consequently the detection of proteins at lower concentrations.

Development of Laser-based IR Transmission Setups for Analysis of Proteins

  

In the last few years, several iterations of EC-QCL based IR transmission setups were developed to analyse the amide I and amide II regions of proteins. The latest iteration provides an improved limit of detection compared to research grade FT-IR instruments at spectra acquisition times lower than 1 min.  

Key Publications:

Beyond FT-IR spectroscopy: EC-QCL based mid-IR transmission spectroscopy of proteins in the amide I and amide II region, Analytical Chemistry, 2018.

External cavity-quantum cascade laser spectroscopy for mid-IR transmission measurements of proteins in aqueous solution, Analytical Chemistry, 2015.

 

Monitoring of perturbation-induced Changes in Protein Secondary Structure

The developed setups were applied to monitor conformational changes in protein secondary structure after different kinds of external perturbation.