recent advancements and applications of ec-qcl based mid ......recently, we applied ec-qcl mid-ir...
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Recent Advancements and Applications of
EC-QCL based mid-IR Transmission Spectroscopy of Proteins
Andreas Schwaighofer1, Mirta R. Alcaráz1,2, Julia Kuligowski3, Héctor Goicoechea2, Bernhard Lendl1
Monitoring Protein Conformational Change Automated Quantum Cascade Laser – based sensor
Sensor setup Introduction
Infrared absorption spectroscopy is a powerful analysis tool to study the secondary structure of proteins. The most prominent absorption band of proteins in the mid-IR spectrum is the
amide I band (1600-1700 cm–1) which is induced by vibrations of the peptide group and allows evaluating the secondary structure. In this spectral region, the most important difficulty of IR
investigations of proteins in aqueous solution is the strong absorbance of the HOH-bending band that overlaps with the amide I band.
For routinely used FTIR spectrometers employing low intensity thermal emitters as light source, suitable path lengths for transmission measurements of proteins in aqueous solution are
restricted to <10 µm. These low optical paths considerable impair the robustness of the system and do not allow flow-through experiments. Recently, we introduced a setup based on a
tunable external-cavity quantum cascade laser (EC-QCL) for mid-IR transmission measurements at high optical paths (38 µm) of the protein amide I band in aqueous solution [1].
This setup has been successfully employed to monitor dynamic changes of protein and polypeptide conformation induced by chemical denaturation and thermal perturbation [2,3]. Most
recently, we applied EC-QCL mid-IR transmission spectroscopy for protein analysis in commercial bovine milk without sample pre-processing [4]. Casein, b-lactoglobulin and total protein content were determined by partial least squares (PLS) modelling after chemometric compensation of the matrix contribution employing science-based calibration (SBC).
[1] Alcaráz, MR; Schwaighofer, A; Kristament, C; Ramer, G; Brandstetter, M; Goicoechea, H; Lendl, B, EC-QC laser spectroscopy for mid-IR transmission measurements of proteins in aqueous solution. Anal. Chem. 2015, 87, 6980-6987.
[2] Alcaráz, MR; Schwaighofer, A; Goicoechea H; Lendl, B; EC-QCL mid-IR transmission spectroscopy for monitoring dynamic changes of protein secondary structure in aqueous solution on the example of b-aggregation in alcohol-denaturated ACT. Anal. Bioanal. Chem. 2016, 408, 3933-3941.
[3] Schwaighofer, A; Alcaraz, MR; Araman, C; Goicoechea, H; Lendl, B; External cavity-quantum cascade laser infrared spectroscopy for secondary structure analysis of proteins at low concentrations. Sci. Rep. 2016, 6, 33556.
[4] Kuligowski, J; Schwaighofer, A; Alcaráz, MR; Quintás, G; Mayer, H; Vento, M; Lendl, B; External cavity-quantum cascade laser (EC-QCL) spectroscopy for protein analysis in bovine milk. Anal. Chim. Acta 2017, 99-105.
Conclusions & Outlook
Experimental Setup
1Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9, A-1060 Vienna, Austria 2Laboratorio de Desarrollo Analítico y Quimiometría, FBCB, Universidad Nacional del Litoral-CONICET, Ciudad Universitaria, 3000 Santa Fe, Argentina
3Neonatal Research Unit, Health Research Institute La Fe, Avenida Fernando Abril Martorell 106, 46026 Valencia, Spain
www.cta.tuwien.ac.at/cavs
Protein Secondary Structure
• Laser-IR transmission measurements were performed in pulsed mode using an
external-cavity quantum cascade laser (Daylight Solutions, USA) with a tuning
range between 1730–1565 cm–1.
• A custom-made temperature-stabilized sample cell enabled IR transmission
measurements at a path length of 38 µm.
• An advanced data processing routine was developed in MatLab employing a
Correlation Optimized Warping (COW) routine. Inherent mode-hop structures of the
laser emission curve are utilized for a rubberband type alignment of consecutive
single beam scans. Scan-to-scan alignment allows for scan averaging and
background-to-sample alignment reduces the noise level in the absorbance
spectrum.
• Wavenumber calibration was performed by referencing the position of the water
vapour absorption bands to band positions taken from a spectra database. This
type of wavenumber calibration accounts for aberrations originating in the light
source and during data acquisition.
Financial support was provided by the Austrian research funding association (FFG) under the scope of the COMET programme within the research project “Industrial Methods for Process Analytical Chemistry - From Measurement Technologies to Information Systems (imPACts)” (contract #843546).
• Dynamic protein conformational change was monitored by QCL-IR spectroscopy
after exposure of a-chymotrypsin to 50% 2,2,2-trifluoroethanol (TFE).
• QCL-IR spectra reveal gradual b-aggregation, indicated by increasing bands at
1621 cm-1 and 1697 cm-1 accompanied by decreasing absorbance at 1655 cm-1.
Spectra were recorded between 2 and 240 min after dissolution in TFE.
• EC QCL-based IR transmission measurements have been successfully employed
to identify characteristic spectral features of proteins with different secondary
structures and to monitor dynamic changes of protein secondary structure.
• Fast and simultaneous determination of casein, b-lactoglobulin and total protein
content in commercial bovine milk samples has been accomplished by QCL-IR
spectroscopy and multivariate analysis without prior sample preparation.
• The potential application of QCL-IR spectroscopy as a fast screening method for
estimating the heat load applied to commercial bovine milk is demonstrated.
• QCL-based IR transmission measurements were successfully employed to identify
characteristic spectral features of proteins with different secondary structures at
protein concentrations as low as 2.5 mg mL-1.
• Protein spectra acquired by EC-QCL transmission measurements show excellent
agreement with absorbance spectra recorded by FTIR spectroscopy .
antiparallel b-sheets
a-helix
Protein Quantification in Bovine Milk
• Partial least squares (PLS) regression models were
employed for quantification of casein, b-lacto-globulin and total protein content in commercial
bovine milk samples.
• QCL-IR spectra of homogenized milk were
recorded without prior sample preparation.
• Science based calibration (SBC) was employed for
compensating the background matrix signal.
• Concentration values obtained by QCL-IR
spectroscopy and multivariate analysis show good
agreement with reference methods (Kjeldahl,
HPLC) requiring multiple sample preparation steps.
• Milk samples experiencing low (pasteurized,
extended shelf life-filtered) and high heat load
(extended shelf life-high temperature short time,
ultra high temperature) were investigated.
• Discrimination between differently processed milk
samples could be accomplished by evaluation of
the b-lactoglobulin concentration.