Infrared absorbance spectroscopy of aqueous proteins: Comparison of transmission and ATR data collection and analysis for secondary structure fitting

Abstract

Attenuated total reflectance (ATR) infrared absorbance spectroscopy of proteins in aqueous solution is much easier to perform than transmission spectroscopy, where short path‐length cells need to be assembled reproducibly. However, the shape of the resulting ATR infrared spectrum varies with the refractive index of the sample and the instrument configuration. Refractive index in turn depends on the absorbance of the sample. In this work, it is shown that a room temperature triglycine sulfate detector and a ZnSe ATR unit can be used to collect reproducible spectra of proteins. A simple method for transforming the protein ATR spectrum into the shape of the transmission spectrum is also given, which proceeds by approximating a Kramers‐Krönig–determined refractive index of water as a sum of four linear components across the amide I and II regions. The light intensity at the crystal surface (with 45° incidence) and its rate of decay away from the surface is determined as a function of the wave number–dependent refractive index as well as the decay of the evanescent wave from the surface. The result is a single correction factor at each wave number. The spectra were normalized to a maximum of 1 between 1600 cm−1 and 1700 cm−1 and a self‐organizing map secondary structure fitting algorithm, SOMSpec, applied using the BioTools reference set. The resulting secondary structure estimates are encouraging for the future of ATR spectroscopy for biopharmaceutical characterization and quality control applications.

The Bouguer‐Beer‐Lambert Law: Shining Light on the Obscure

“…This means that the absorbance band maxima are redshifted towards the maxima of the index of refraction and that the band shapes can strongly change. [52][53][54][55] A simple way to reduce this problem is to use high index ATR crystal materials like Si and Ge and to apply high angles of incidence. Unfortunately, this means that the penetration depths and, thereby, the overall intensities are reduced and by that the sensitivity of the method.…”

ATR-FTIR spectroscopy and spectroscopic imaging for the analysis of biopharmaceuticals

“…If proteins are of a high concentration (N30 mg/ml), which is typical in biopharmaceutical production, amide bands can more easily be seen, however lower concentrations also yield good Amide I and II spectra ( Fig. 4) [63]. Macro ATR-FTIR spectroscopy is commonly used due to the ease of sample preparation and data collection.…”

“…When properly utilised, FTIR spectroscopy can replace some other, more traditional methods [85]. Although circular dichroism (CD) can be used to estimate secondary structure, it is unsuitable for highly concentrated samples, or samples containing other highly absorbing molecules such as excipients [63]. In contrast, FTIR spectroscopy, particularly ATR-FTIR spectroscopy, caters to these requirements.…”

Searching for universal model of amyloid signaling motifs using probabilistic context-free grammars

“…Amyloid-like aggregation To determine aggregation properties of studied peptides Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) experiments were carried out. Vibrational spectroscopy is widely used for protein and polypeptides secondary structure analysis [ 69 , 70 ] and for monitoring the aggregation processes in amyloids studies [ 71 – 73 ]. The Amide I band (1700–1600 cm ) corresponding to C=O stretching vibrations and the Amide II band (1600–1500 cm ) arising mainly from in-plane N–H bending of the peptide bonds are the most useful for secondary structure estimation.…”