Chapter 18 Fourier Transform Infrared Spectroscopy of Peptides Kunal Bakshi, Mangala R. Liyanage, David B. Volkin, and C. Russell Middaugh Abstract Fourier transform infrared (FTIR) spectroscopy provides data that are widely used for secondary structure characterization of peptides. A wide array of available sampling methods permits structural analysis of peptides in diverse environments such as aqueous solution (including optically turbid media), powders, detergent micelles, and lipid bilayers. In some cases, side chain vibrations can also be resolved and used for tertiary structure and chemical analysis. Data from several low-resolution spectroscopic techniques, including FTIR, can be combined to generate an empirical phase diagram, an overall picture of peptide structure as a function of environmental conditions that can aid in the global interpretation of large amounts of spectroscopic data. Key words Fourier transform infrared spectroscopy, Peptide, Empirical phase diagram
1
Introduction Fourier transform infrared (FTIR) spectroscopy is increasingly becoming a method of choice to characterize and study structural changes in peptides. The ready availability of a wide array of sampling methods based on transmission and reflection principles allows FTIR spectroscopy to circumvent sample-related constraints often encountered by structural methods such as NMR, X-ray crystallography, and circular dichroism. These sampling methods permit structural analysis of peptides in diverse environments such as aqueous solution, powders, detergent micelles, and lipid bilayers. Due to the use of long-wavelength radiation that minimizes scattering problems, FTIR measurements can also be performed in optically turbid media, a common problem often encountered during the study of peptides. Additionally, the high speed of data acquisition, high signal-to-noise ratio, and low cost
Kunal Bakshi and Mangala R. Liyanage have contributed equally to this chapter. Andrew E. Nixon (ed.), Therapeutic Peptides: Methods and Protocols, Methods in Molecular Biology, vol. 1088, DOI 10.1007/978-1-62703-673-3_18, © Springer Science+Business Media, LLC 2014
255
256
Kunal Bakshi et al.
of instrumentation make FTIR spectroscopy a highly desirable method for secondary structure characterization of peptides as described below.
2
Materials 1. A research-grade FTIR instrument: Some of the principal manufacturers are Perkin-Elmer (Waltham, MA), Jasco (Easton, MD), Bruker Optics (Billerica, MA), and Varian (Palo Alto, CA). The typical FTIR spectrometer requirements for peptide analysis include moderate resolution (0.5–4 cm−1) combined with a high-sensitivity nitrogen-cooled mercury–cadmium–telluride (Hg–Cd–Te) detector. A diverse variety of sampling geometries and assemblies are available and the reader should refer to individual manufacturer’s manuals for further details. Three of the most commonly used sampling techniques for peptide samples are transmittance, attenuated total reflectance, and diffuse reflectance and are summarized below. 2. Transmittance: This approach is the most conventional sampling method. Cells are constructed from two IR transparent windows with a spacer in between to define the path length. A number of cell options are available that include sealed cells, demountable cells with a choice of different IR window materials (CaF2, KBr) depending on sample compatibility, and different spacers for different path lengths. A good choice of window material is calcium fluoride, which is transparent in the region 1,000–5,000 cm−1 and insoluble in water. Both tin and Teflon spacers are available. Solid peptides can also be pressed in the presence of KBr into IR transparent discs for spectral analysis. 3. Attenuated total reflectance (ATR): One form of ATR sampling consists of a horizontal plate commonly made of zinc selenide (ZnSe), germanium (Ge), or diamond crystals. The sample is loaded on top of the horizontal plate and the IR light is passed through the bottom of this high-refractive-index material such that an evanescent wave is produced that penetrates the sample. This sampling method allows samples in a variety of physical states (solution, suspension, films, and gels) to be probed with ease. A major drawback of this technique is that peptide samples may be adsorbed on the surface of the ATR unit and thus the spectral features may be altered. Furthermore, the observed spectrum consists primarily of material near the surface itself. 4. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS): Spectra of powdered samples can be recorded with no or minimal sample preparation using this geometry. Powder can be directly placed into a sample cup or on the top of an
Fourier Transform Infrared Spectroscopy of Peptides
257
infrared inert material such as KBr, which is placed in the sample cup. The DRIFTS cell contains mirrors that direct the beam onto the surface of the powder sample. The mirrors may need to be aligned by following the manufacturer’s instructions. Spectra must be corrected for the dependence of the spectra on wavelength by a procedure known as the Kubelka– Munk correction, which is typically available with the instrument’s software. 5. In addition to these more common techniques, a broad range of accessories such as photoacoustic modalities are also available to overcome sample-related limitations. 6. Standard supplies to be used with FTIR experiments should always be of highest quality available to avoid contamination problems.
3
Methods
3.1 Preparation of the Instrument
1. The instrument should be turned on and allowed to stabilize for at least 60 min before collecting spectra. The instrument and the sample cell should also be allowed to equilibrate for 15–20 min at the set temperature. 2. The instrument should always be purged with dry nitrogen or dry air at a pressure between 20 and 40 psi. The detector should be cooled by filling the detector-cooling system with liquid nitrogen. Deuterated triglycine sulfate (DGTS) or other detectors can also be used but they are less sensitive. 3. Any purge shutters should be closed whenever the sample compartment lid is open to minimize water vapor and CO2 in the optical system. Modern instruments are often designed in such a way to avoid exposure of the sample compartment to the air. 4. Prior to data collection, the interferogram or single-beam spectrum should be checked for energy throughput and spectral quality with an empty cell. 5. The transmittance cell must be thoroughly cleaned immediately after use, as instructed by the supplier. Generally, this involves removing the sample with a tissue, followed by several solvent washes and a final ethanol wash. The use of a polishing kit to further polish the window surface may also be necessary. Wear gloves to prevent fogging. The cleaned surface should be clear and free from scratches. 6. ATR crystals are cleaned by gently clearing the sample surface using a lint-free tissue soaked in solvent. Peptide samples can be removed by using 10 % SDS solution, followed by wash steps with water (repeated four to five times), and a final step with isopropanol.
258
Kunal Bakshi et al.
3.2 Sample Preparation
1. FTIR sample preparation is dependent upon the sampling technique to be employed during spectral acquisition. For transmission spectroscopy, peptide solutions in H2O or D2O can be directly loaded into liquid transmittance or ATR cells. In dried form, peptides can be prepared as KBr pellets or placed directly in DRIFTS cells. 2. Liquid samples are spread as thin, even films to maintain a constant path length. For demountable cells, the sample is placed on one plate and the second plate is placed on top, with circular motions to form an even film. Modern liquid transmittance cells possess one or two loading/unloading ports with appropriate parts. 3. For aqueous samples, due to strong interfering absorbance bands in the amide I region (1,600–1,700 cm−1), shorter path length (2–10 μm) cells and higher peptide concentrations (~2– 10 mg/ml) have traditionally been employed. Recently, the increased sensitivity of spectrometers combined with ATR cells has permitted much lower aqueous peptide concentrations (
1
Introduction Fourier transform infrared (FTIR) spectroscopy is increasingly becoming a method of choice to characterize and study structural changes in peptides. The ready availability of a wide array of sampling methods based on transmission and reflection principles allows FTIR spectroscopy to circumvent sample-related constraints often encountered by structural methods such as NMR, X-ray crystallography, and circular dichroism. These sampling methods permit structural analysis of peptides in diverse environments such as aqueous solution, powders, detergent micelles, and lipid bilayers. Due to the use of long-wavelength radiation that minimizes scattering problems, FTIR measurements can also be performed in optically turbid media, a common problem often encountered during the study of peptides. Additionally, the high speed of data acquisition, high signal-to-noise ratio, and low cost
Kunal Bakshi and Mangala R. Liyanage have contributed equally to this chapter. Andrew E. Nixon (ed.), Therapeutic Peptides: Methods and Protocols, Methods in Molecular Biology, vol. 1088, DOI 10.1007/978-1-62703-673-3_18, © Springer Science+Business Media, LLC 2014
255
256
Kunal Bakshi et al.
of instrumentation make FTIR spectroscopy a highly desirable method for secondary structure characterization of peptides as described below.
2
Materials 1. A research-grade FTIR instrument: Some of the principal manufacturers are Perkin-Elmer (Waltham, MA), Jasco (Easton, MD), Bruker Optics (Billerica, MA), and Varian (Palo Alto, CA). The typical FTIR spectrometer requirements for peptide analysis include moderate resolution (0.5–4 cm−1) combined with a high-sensitivity nitrogen-cooled mercury–cadmium–telluride (Hg–Cd–Te) detector. A diverse variety of sampling geometries and assemblies are available and the reader should refer to individual manufacturer’s manuals for further details. Three of the most commonly used sampling techniques for peptide samples are transmittance, attenuated total reflectance, and diffuse reflectance and are summarized below. 2. Transmittance: This approach is the most conventional sampling method. Cells are constructed from two IR transparent windows with a spacer in between to define the path length. A number of cell options are available that include sealed cells, demountable cells with a choice of different IR window materials (CaF2, KBr) depending on sample compatibility, and different spacers for different path lengths. A good choice of window material is calcium fluoride, which is transparent in the region 1,000–5,000 cm−1 and insoluble in water. Both tin and Teflon spacers are available. Solid peptides can also be pressed in the presence of KBr into IR transparent discs for spectral analysis. 3. Attenuated total reflectance (ATR): One form of ATR sampling consists of a horizontal plate commonly made of zinc selenide (ZnSe), germanium (Ge), or diamond crystals. The sample is loaded on top of the horizontal plate and the IR light is passed through the bottom of this high-refractive-index material such that an evanescent wave is produced that penetrates the sample. This sampling method allows samples in a variety of physical states (solution, suspension, films, and gels) to be probed with ease. A major drawback of this technique is that peptide samples may be adsorbed on the surface of the ATR unit and thus the spectral features may be altered. Furthermore, the observed spectrum consists primarily of material near the surface itself. 4. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS): Spectra of powdered samples can be recorded with no or minimal sample preparation using this geometry. Powder can be directly placed into a sample cup or on the top of an
Fourier Transform Infrared Spectroscopy of Peptides
257
infrared inert material such as KBr, which is placed in the sample cup. The DRIFTS cell contains mirrors that direct the beam onto the surface of the powder sample. The mirrors may need to be aligned by following the manufacturer’s instructions. Spectra must be corrected for the dependence of the spectra on wavelength by a procedure known as the Kubelka– Munk correction, which is typically available with the instrument’s software. 5. In addition to these more common techniques, a broad range of accessories such as photoacoustic modalities are also available to overcome sample-related limitations. 6. Standard supplies to be used with FTIR experiments should always be of highest quality available to avoid contamination problems.
3
Methods
3.1 Preparation of the Instrument
1. The instrument should be turned on and allowed to stabilize for at least 60 min before collecting spectra. The instrument and the sample cell should also be allowed to equilibrate for 15–20 min at the set temperature. 2. The instrument should always be purged with dry nitrogen or dry air at a pressure between 20 and 40 psi. The detector should be cooled by filling the detector-cooling system with liquid nitrogen. Deuterated triglycine sulfate (DGTS) or other detectors can also be used but they are less sensitive. 3. Any purge shutters should be closed whenever the sample compartment lid is open to minimize water vapor and CO2 in the optical system. Modern instruments are often designed in such a way to avoid exposure of the sample compartment to the air. 4. Prior to data collection, the interferogram or single-beam spectrum should be checked for energy throughput and spectral quality with an empty cell. 5. The transmittance cell must be thoroughly cleaned immediately after use, as instructed by the supplier. Generally, this involves removing the sample with a tissue, followed by several solvent washes and a final ethanol wash. The use of a polishing kit to further polish the window surface may also be necessary. Wear gloves to prevent fogging. The cleaned surface should be clear and free from scratches. 6. ATR crystals are cleaned by gently clearing the sample surface using a lint-free tissue soaked in solvent. Peptide samples can be removed by using 10 % SDS solution, followed by wash steps with water (repeated four to five times), and a final step with isopropanol.
258
Kunal Bakshi et al.
3.2 Sample Preparation
1. FTIR sample preparation is dependent upon the sampling technique to be employed during spectral acquisition. For transmission spectroscopy, peptide solutions in H2O or D2O can be directly loaded into liquid transmittance or ATR cells. In dried form, peptides can be prepared as KBr pellets or placed directly in DRIFTS cells. 2. Liquid samples are spread as thin, even films to maintain a constant path length. For demountable cells, the sample is placed on one plate and the second plate is placed on top, with circular motions to form an even film. Modern liquid transmittance cells possess one or two loading/unloading ports with appropriate parts. 3. For aqueous samples, due to strong interfering absorbance bands in the amide I region (1,600–1,700 cm−1), shorter path length (2–10 μm) cells and higher peptide concentrations (~2– 10 mg/ml) have traditionally been employed. Recently, the increased sensitivity of spectrometers combined with ATR cells has permitted much lower aqueous peptide concentrations (
