# Peak Fitting Applied to Fourier Transform Infrared and Raman Spectroscopic Analysis of Proteins
## 1 Introduction  
Infrared spectroscopy is one of the oldest techniques to study the secondary structure of macropeptides and proteins [1; 2; 3; 4; 5]. Alternative techniques such as circular dichroism (CD) and X-ray crystallography have also been utilized to gain information about protein structure; X-ray crystallography relies on the protein's ability to crystallize [6]. CD spectroscopy is a technique based on the absorption of light and measures the difference in absorbance of left and right circularly polarised light [7; 8; 9]. CD will require protein extraction prior to analysis and will, hence, not allow the in situ study of proteins (i.e., gluten) in a range of complex food systems, such as dough and baked products. However, FTIR and FT-Raman techniques have the ability to study the proteins in the complex food matrix without the need for extensive sample preparation or extraction [10]. Nuclear magnetic resonance spectroscopy can also be used to study protein structure in solution but the processing of data obtained for large proteins is a very complicated process [6]. Infrared and Raman spectroscopy, on the other hand, are non-invasive vibrational spectroscopy techniques and can be used to study the protein structure in the native state and in a wide range of different environments [6]. For instance, FTIR has been used to study the changes in gluten secondary structure during dough mixing [11; 12; 13; 14], enzymatic modification [15], hydration [16; 17], and upon bran addition to a viscoelastic gluten dough [18]. Protein-containing samples display nine characteristic vibrational absorption bands in an IR spectrum (~200-3300 cm\({}^{-1}\)) which arise from different vibrational modes of the amide groups of proteins (found in the peptide bonds), namely amide A, B, and I-VII [19]. Among these, the amideI band which mainly probes stretching vibrations of the C=O bonds in the peptide backbone has been proven to be the most sensitive and popular band to study protein secondary structure [5; 19]. Each type of secondary structure correlates to a slightly different C=O stretching frequency in the amide I band region of the spectrum due to its unique molecular geometry and hydrogen bond pattern [6; 19]. Amide II also provides information on the vibrational bands of the protein backbone. However, it derives mostly from in-plane N-H bending (40-60% of the potential energy), and C-N stretching (18-40%), resulting in less sensitivity and specificity for protein conformational changes compared to the amide I band [19]. When studying aqueous solutions of proteins, strong interference of intense infrared absorption of water often distorts the IR spectra, complicating the practical use of FTIR in high moisture systems [10]. The H-O-H scissoring/bending vibration shows a strong absorbance in the amide I band region, which makes the analysis of this band difficult. A common approach is subtracting the reference spectrum of the pure solvent, i.e., water. There has been a number of studies for proteins solubilized in H\({}_{2}\)O in which a reference H\({}_{2}\)O spectrum was subtracted from the sample spectrum [5; 20; 21; 22]. Other strategies have also been explored and utilized thus far to solve this issue, such as using D\({}_{2}\)O as a solvent instead of H\({}_{2}\)O [1; 2; 23; 24]. However, the use of D\({}_{2}\)O results in very different solvent properties relative to H\({}_{2}\)O and it is known to affect hydrogen bond dynamics [6; 25; 26]. The different solvent properties of D\({}_{2}\)O compared with H\({}_{2}\)O can alter the structure of proteins by changing the balance between intra- and intermolecular hydrogen bonds and hydration interactions [26]. Overall, an ideal subtraction needs the complete overlay of both the reference water spectrum and the sample water spectrum [27]. As the interaction of molecules (i.e., proteins) with water can cause small shifts in the H\({}_{2}\)O bending signal, there is a risk of creating subtraction artefacts.