Biology Assays & Protocols
- Protein purification
- Protein fibrils
- Conformational stability assay
- BCA protein assay
Fourier transform infrared (FTIR) spectroscopy assay
Fourier Transform Infrared Spectroscopy (FTIR)
Fourier transform infrared (FTIR) spectroscopy is used to detect the presence of β-sheet secondary structure. Typically, the sample is examined before and after aggregation and an increase in β-sheet secondary structure is observed upon amyloid fibril formation. For FTIR analysis, sample preparation is very important. First, the ion pairing agent used in the purification of the peptide or protein needs to be IR compatible. Commercial peptides generally have TFA as the ion pair but TFA has an infrared vibration that interferes with the analysis of protein/peptide secondary structure and, therefore, must be exchanged to another ion pair. Next, the residual water adsorbed to the peptide or protein needs to be exchanged to D2O. This step is not designed to fully deuterate the protein/peptide because, if the peptide or protein is subjected to conditions to fully deuterate the sample, this may lead to a different FTIR spectrum by prematurely inducing aggregation. The peptide or protein is then analyzed by FTIR in D2O and a spectrum recorded; this procedure is repeated after fibril formation. The choice of sample holder and scanning parameters for FTIR analysis varies, but a dismountable sample cell (which can be thoroughly and easily cleaned) and a minimum of 64 scans encompassing the range of 1500–1800 cm-1 is ideal. The amide I band, which is used to assign secondary structure, occurs in the region between 1600 and 1700 cm-1. However, many interesting side chain vibrations occur on the edge of this range and detection between 1500 and 1800 cm-1 will allow these vibrations to be observed. The most common detectors are DTGS and MCT. DTGS is particularly stable and, therefore, provides superior water vapor subtraction while the MCT detector is more sensitive but generally less stable and needs to be cooled with LN2.
Procedure for FTIR analysis of amyloid fibrils:
1. Exchange the ion pairing agent: dissolve the peptide/protein in 5mM HCl, freeze in liquid nitrogen (LN2), lyophilize, and repeat this process 4–5 times. Alternatively, the peptide/protein can be repurified by RP-HPLC using HCl instead of TFA in the buffers.
2. Exchange the residual H2O to D2O: dissolve the peptide in a small amount of D2O, incubate at room temperature for 30 min, freeze (LN2), and lyophilize.
3. Amyloid fibrils should be formed in D2O with the pD adjusted with DCl/NaOD. As a general rule, it is difficult to get quality FTIR spectra from fibrils formed in H2O and then exchanged to D2O.
4a. Data acquisition in D2O: purge sample chamber for 20 min, take a scan of the empty chamber (as a background), purge for 20 min, take a scan of the same D2O solution used for sample preparation, purge for 20 min, take a scan of the sample, and subtract the D2O spectrum from the sample spectrum*.
4b. Data Acquisition in the solid state: purge sample chamber for 20 min, take a scan of the empty chamber (as a background), purge for 20 min, take a scan of the empty sample cell, purge for 20 min, dry the peptide or fibril sample onto the sample cell window using a gentle stream of nitrogen and take a scan of the sample, and subtract the D2O spectrum from the sample spectrum*.
5. Examine the Amide I region (1600–1700 cm-1) of the sample spectrum. β-sheet secondary structure is detected by the presence of a band near 1620 cm-1.
6. Note: FTIR can be performed in H2O instead of D2O, but very high sample concentrations are required and the high sample concentration can make it difficult to obtain a sample spectrum of the monomeric peptide/protein.
*Some FTIR instruments do not require purging to eliminate water vapor (e.g., Perkin Elmer Spectrum One).