Fourier transform infrared (FTIR) spectroscopy
Coriolis Pharma is specialized in providing contract laboratory services in protein analysis, including Fourier transform infrared (FTIR) spectroscopy.
FTIR spectroscopy can be applied to monitor protein structure in the liquid and dried, e.g. lyophilized state. Therefore, it is a valuable tool used during the development of lyophilized formulations.
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 1700-1500 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 1700-1600 cm-1 and 1600-1500 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. Measurements can be obtained for protein samples in solution, but a high (>5 mg/mL) protein concentration is required. Both liquid (Transmission, ATR) and solid (ATR) samples can be analyzed at Coriolis Pharma.