Fourier transform infrared (FTIR) spectroscopy
Fourier transform infrared (FTIR) spectroscopy is a highly valuable technique to monitor protein structure in the liquid and dried, e.g., lyophilized state.
FTIR spectra can be used to identify a wide range of compounds by comparing the measured spectra to spectral databases. For proteins, FTIR spectra from wavenumbers 1,700-1,500 cm-1 can be used to determine structural properties. Measuring protein absorbance over these wavenumbers gives two absorption bands, conventionally called Amide I and Amide II and lying between wavenumbers 1,700 - 1,600 cm-1 and 1,600 - 1,500 cm-1, respectively.
The Amide I band is due to C=O stretching vibrations of the peptide bonds, which are modulated by the secondary structure (α-helix, β-sheet, etc.). Secondary structural content can be obtained by comparing the measured spectra to the spectra obtained for proteins with known secondary structures.
The Amide II band is due to C-N stretching vibrations in combination with N-H bending. Amide II absorbance can be used, for instance, to report on protein unfolding based on the extent of hydrogen (H) exchanged for deuterium (D) in H-D exchange experiments.
Water can interfere with FTIR measurements of protein samples, because it strongly absorbs in the Amide I region. Consequently, FTIR is best suited for lyophilized (freeze-dried) protein samples. Nevertheless, measurements can be obtained for protein samples in solution, but a relatively high (typically >5 mg/mL) protein concentration is required. Both liquid (transmission, attenuated total reflection (ATR)) and solid (ATR) samples can be analyzed at Coriolis Pharma.
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