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:

• Broadband laser-based mid-infrared spectroscopy employing a quantum cascade detector for milk protein analysis, Sensors and Actuators B-Chemical, 2022.
• The next Generation of IR Spectroscopy: EC-QCL based mid-IR Transmission Spectroscopy of Proteins with Balanced Detection, Analytical Chemistry, 2020.
• 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.

Book Chapter:

• Quantum cascade laser-based infrared transmission spectroscopy of proteins in solution, Vibrational Spectroscopy in Protein Research, 2020.

Inline Monitoring of Proteins from Preparative Liquid Chromatography

A commercial EC-QCL based IR Instrument (Chemdetect Analyzer by Daylight Solutions) was employed for inline monitoring of proteins from preparative ion-exchange chromatography (IEX) and size exclusion spectroscopy (SEC).

Key Publications:

• QCL-IR Spectroscopy for In-Line Monitoring of Proteins from Preparative Ion-Exchange Chromatography, Analytical Chemistry, 2022.
• Laser-based mid-infrared spectroscopy enables in-line detection of protein secondary structure from preparative liquid chromatography, SPIE Proceedings, 2022.
• Broadband laser-based mid-IR spectroscopy for analysis of proteins and monitoring of enzyme activity, Spectrochimica Acta Part A, 2021.

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.  

Key Publications:

pH titration of β-lactoglobulin monitored by laser-based Mid-IR transmission spectroscopy coupled to chemometric analysis, Spectrochimica Acta A, 2020., opens an external URL in a new window

External cavity-quantum cascade laser infrared spectroscopy for secondary structure analysis of proteins at low concentrations, Nature Scientific Reports, 2016., opens an external URL in a new window

EC-QCL mid-IR transmission spectroscopy for monitoring dynamic changes of protein secondary structure in aqueous solution on the example of beta-aggregation in alcohol-denaturated alpha-chymotrypsin, Analytical Bioanalytical Chemistry, 2016., opens an external URL in a new window

Quantitation of Individual Proteins in Bovine Milk Samples  

QCL-IR spectroscopy was employed for quantitation of major proteins present in commercial bovine milk. Evaluation of the concentration levels of the temperature sensitive proteins beta-lactoglobulin and alpha-lactalbumin enables the discrimination between different milk types.  

Key Publications:

• High-throughput quantitation of bovine milk proteins and discrimination of commercial milk types by external cavity-quantum cascade laser spectroscopy and chemometrics, Analyst, 2019.
• Fast quantification of bovine milk proteins employing external cavity-quantum cascade laser spectroscopy, Food Chemistry, 2018.
• External cavity-quantum cascade laser (EC-QCL) spectroscopy for protein analysis in bovine milk, Analytica Chimica Acta, 2017.  

Researchers

Shilpa Vijayakumar

Alicja Dabrowska

Georg Ramer