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Beyond FT-IR Spectroscopy: EC-QCL based mid-IR Transmission Spectroscopy of Proteins in the Amide I and Amide II Region Andreas Schwaighofer, Milagros Montemurro, Stephan Freitag, Christian Kristament, María Julia Culzoni, and Bernhard Lendl Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01632 • Publication Date (Web): 15 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018
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Analytical Chemistry
Beyond FT-IR Spectroscopy: EC-QCL based mid-IR Transmission Spectroscopy of Proteins in the Amide I and Amide II Region Andreas Schwaighofer,†,‡ Milagros Montemurro, †,§,‡ Stephan Freitag,† Christian Kristament,† María J. Culzoni,§ Bernhard Lendl*,† †
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-UPA, 1060 Vienna, Austria § Laboratorio de Desarrollo Analítico y Quimiometría (LADAQ), Cátedra de Química Analítica I, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral-CONICET, Ciudad Universitaria, 3000 Santa Fe, Argentina *Corresponding author E-mail:
[email protected] 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 approx. 2 compared to research-grade 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-1. Mathematical evaluation of the spectral overlap confirms excellent agreement of the 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.
1645 cm-1, that overlaps with the protein amide I band. Thus, applicable path lengths are restricted to 10 mg mL−1) are required, as these low path lengths limit the intensities of the IR bands and the signal-tonoise ratio (SNR) at a given concentration.5 Furthermore, experimental challenges in cell and sample handling arise due to the low flow cross section that lead to a large pressure drop in the cell and vulnerability to cell clogging. More than two decades ago, first quantum cascade lasers (QCLs) were introduced as a new light source for the mid-IR region that provide polarized and coherent light with spectral power densities several orders of magnitude higher than thermal light.12 They are unipolar lasers based on intersubband transitions of electrons within the semiconductors conduction band. Due to the limited tuning range of only a few wavenumbers, distributed feedback (DFB) lasers were predominantly employed for gas phase analysis.13 Ten years ago, a new type of these mid-IR lasers, external-cavity QCL (ECQCLs) became commercially available, which combine high emission powers with spectral tuning ranges of several hundred wavenumbers. The high available emission powers enabled to employ 4-5 times larger path lengths for transmission
Fourier transform infrared (FT-IR) spectroscopy is a wellestablished and powerful technique for analysis of the structure and dynamics of polypeptides and proteins.1-3 Vibrations of the polypeptide repeat units of proteins result in nine characteristic group frequencies in the mid-IR region referred to as amide bands. The differing pattern of hydrogen bonding, dipole–dipole interactions and geometric orientations in the αhelices, β-sheets, turns and random coil structures induce different frequencies of the C=O and N-H vibrations that can be correlated with the respective secondary structural folding.4 The amide I band (1700–1600 cm−1) originating from the C=O stretching and N-H in-phase bending vibration of the amide group has been recognised to be most sensitive to secondary structure.5,6 Furthermore, also the amide II band (1600– 1500 cm−1) arising from N–H bending and C–N stretching vibrations was shown to be sensitive to protein structure.7 It has been shown that additional and more in-depth information about protein secondary structure can be gained by collective analysis of both spectral regions, particularly with chemometric analysis.8-10 IR transmission measurements are frequently employed for studies of protein structure in solution. A limitation when working with proteins in aqueous solution is the strong absorbance of the HOH bending vibration of water centered at 1
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measurements of glucose14-16 and proteins17-22 than achievable with conventional FT-IR spectrometers. Furthermore, EC-QCLs were employed for broadband IR transmission measurements to study protein dynamics by fast kinetic experiments at high temporal resolution.23-25 IR measurements of diverse liquid samples were performed using waveguides26,27 and the ATR28 technique. In addition, a method was presented for indirect absorbance detection in the form of an EC-QCL based photothermal phase shift interferometer.29 Apart from absorption measurements, the polarized nature of the emitted light was utilized for vibrational circular dischroism (VCD) measurements.30-32 Further, the coherence of the emitted QCL light enabled to develop a Mach–Zehnder interferometer to simultaneously record absorption and dispersion spectra of liquid samples.33 These manifold examples demonstrate that the unique properties of EC-QCLs led to the implementation of novel experimental approaches and applications in different fields.34 However so far, their basic advantage of the provided high emission power could not effectively be translated to significantly higher signal-to-noise ratios than in FT-IR spectroscopy in the case of broadband transmission spectroscopy. FT-IR spectroscopy is currently considered as the gold standard due to its broad spectral range covering the entire mid-IR region, excellent SNR and, finally, the absolute wavenumber accuracy, originating from the precise interferometer control by the HeNe laser interferogram. This wavenumber accuracy facilitates scan averaging in order to achieve the notoriously low noise levels. In FT-IR spectroscopy, the final signal noise is determined by the detector noise, due to the low noise levels of the low power thermal light sources. The detected signal contains both the optical signal and the noise, which has its own frequency spectrum, and consequently the noise power is distributed over the complete spectral range by the Fourier transformation.35 A pronounced drawback for 1st generation EC-QCL light sources are the mechanical imperfections of the external cavity. Triggering inaccuracies and imprecisions throughout the motion of the grating introduce a constant and variable spectral mismatch in the fine structure of consecutive scans in the time (i.e. wavenumber) axis of the emitted spectrum. This small deviation of only approx. 0.5 cm-1 in the single beam spectrum (i) prevents efficient scan averaging for noise reduction and (ii) introduces significant noise in the corresponding absorbance spectrum. Consequently, processing routines of the measured raw data were implemented to reduce the effects of this spectral mismatch. Initially, shifting of the recorded scans along the time axis was used to achieve maximum alignment, followed by considerable filtering.36 Later, a routine for noise reduction was devised based on correlation optimized warping of successive scans to correct for non-constant shifts in the fine structure throughout the spectrum.17 A further impediment for accomplishing similar signal-to-noise ratios as with FT-IR spectrometers within reasonable time was the limited sweep velocity in the range of 100 cm-1 s-1 in early commercially available EC-QCLs. Using latest generation EC-QCLs, tuning rates of up to 25000 cm-1 s-1 (LaserTune, Block Engineering) and 5000 cm-1 s-1 (MIRcat-QT, Daylight Solutions) can been reached. Furthermore, scanning speeds of up to 600000 cm-1 s1 have been published employing a resonantly driven microopto-electro-mechanical systems (MOEMS) grating.37,38
In this work, we present IR transmission measurements of proteins in aqueous solutions in the amide I and amide II region, employing a latest generation EC-QCL. A dataprocessing routine is presented for noise reduction, which discards occasional EC-QCL scans that are shifted in the wavenumber axis. The performance of the setup regarding SNR was compared to research-grade FT-IR spectrometers at equal acquisition times. The shape of the obtained IR spectra of proteins with different secondary structures were contrasted to FT-IR spectra and showed excellent comparability.
EXPERIMENTAL SECTION Reagents and Samples. Sodium phosphate monobasic dihydrate p.a. (NaH2PO4•2H2O) was purchased from Fluka (Buchs, Switzerland), sodium phosphate dibasic dihydrate (Na2HPO4•2H2O) BioUltra, for molecular biology, and sodium hydroxide solution 50% in water, were obtained from SigmaAldrich (Steinheim, Germany). β-Lactoglobulin from bovine milk (≥85%), bovine serum albumin (≥98%) and lysozyme from chicken egg white (>90%) were obtained by SigmaAldrich (Steinheim, Germany) and used as purchased. Appropriate amount of lyophilized protein powder was dissolved in 16.0 mmol L–1 phosphate buffer pH 7.0. Ultrapure water (18 MΩ) was used for preparation of all solutions, obtained with a Milli-Q water purification system from Millipore (Bedford, USA). FT-IR Measurements. FT-IR absorption measurements were performed using a Bruker Vertex 80v FT-IR spectrometer (Ettlingen, Germany) equipped with a liquid nitrogen cooled HgCdTe (mercury cadmium telluride) detector (D*= 4 × 1010 cm Hz0.5 W–1 at 9.2 µm) and a Bruker Tensor 30 FT-IR spectrometer equipped with a DLaTGS (deuterated lanthanum αalanine doped triglycine sulfate) detector (D*= 6 × 108 cm Hz0.5 W–1 at 9.2 µm). The samples were placed between two CaF2 windows separated by an 8 µm-thick spacer. During measurements, the spectrometer was flushed with dry air for at least 5 minutes prior to spectrum acquisition. Spectra were acquired with a spectral resolution of 2 cm–1 in double-sided acquisition mode. A total of 81 (Vertex operated at 80 kHz scanner velocity) and 21 (Tensor operated at 20 kHz scanner velocity) scans were averaged per spectrum (acquisition time: 53 s), which was calculated using a Blackman-Harris 3-term apodization function and a zero filling factor of 2. All spectra were acquired at 25 °C. Spectra were analyzed using the software package OPUS 7.2 (Bruker, Ettlingen, Germany). If necessary, absorption bands of water vapor in the atmosphere were subtracted. Experimental Setup for QCL Measurements. The experimental setup is shown in Figure 1. For the measurements, 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, resulting in a duty cycle of 50%. The manufacturer states laser power variations of
