# Peak Fitting Applied to Fourier Transform Infrared and Raman Spectroscopic Analysis of Proteins
## 4 Discussion  
As mentioned earlier, the amide I band in FTIR spectra is mainly composed of C=O stretching (around 80%) [37] and, to a lesser extent, NH bending vibrations. The exact location of these vibrations depends on the hydrogen bonds these C=O form with proximate NH groups [19]. The secondary structure of a protein determines with which NH group hydrogen bonds will be formed, turning the vibrational spectra into fingerprints of the secondary structure [6]. The secondary structure of a protein backbone is considered as the linear sum of some fundamental secondary structural elements (\(\alpha\)-helix, \(\beta\)-sheet, \(\beta\)-turn, and random coil) [6]. The percentage of each secondary structure is only related to their spectral intensity since the molar absorptivity of C=O stretching vibration for all secondary structural elements is essentially the same [6]. As stated above, the secondary structure of four standard proteins was investigated by both FTIR and FT-Raman. These proteins were chosen because of their known three-dimensional structures that have been extensively studied by circular dichroism [8; 9], FTIR [5], Raman spectroscopy [47], and X-ray crystallography [42; 48]. Inspection of the FTIR spectra showed that while the amide I band shows the same peak position in original spectra, the band shapes were quite different after water subtraction (Figure 1). This shows the necessity of the water subtraction for FTIR spectra since the broad and intense peak of water overlapping with the amide I region makes the protein fingerprint peaks difficult to distinguish [27]. Conversely, FT-Raman spectra were not subjected to water subtraction due to the weak signal of OH stretching vibration (of water) in the Raman amide I region [29]. Curve fitting was done (Figure 3) to analyse the FTIR spectra in a quantitative way. The relative amounts of each secondary structure of the four proteins calculated as outlined above were consistent with the relative amounts reported in previous FTIR [5], circular dichroism [9], and X-ray crystallography [42] studies. Concanavalin A is often used as a reference protein for FTIR analysis since it is known for its high content of \(\beta\)-sheet structure. The curve-fitted spectrum of concanavalin A showed a peak maximum around 1636 cm\({}^{-1}\) corresponding to the antiparallel intramolecular \(\beta\)-sheet conformation which is the main secondary structural component in concanavalin A [37; 49]. An intense band was also observed at 1626 cm\({}^{-1}\), which is associated to the intermolecular \(\beta\)-sheets with stronger hydrogen bonds. It has been proposed that this band comes from the so-called \"distorted \(\beta\)-type structures\" that are formed by peptide residues located at the end of the main strands of the \(\beta\)-sheets [37]. The band around 1618 cm\({}^{-1}\) is also attributed to intermolecular \(\beta\)-sheets with strong H-bonds [49]. It is worthy to note in this context that a stronger hydrogen bond will lead to a lower C=O stretching frequency [50]. The band assignment of protein secondary structures is further complicated by absorption of amino acid side chains in the protein structures [38]. This absorption is superimposed on the amide I and II bands absorption (Table 3) and contributes 10 to 20% of the overall absorption of globular proteins in the amide I and II regions [6; 20; 21]. Aromatic side chains of amino acids also contribute to the FT-Raman amide I and II regions. In particular, tyrosine, phenylalanine, and tryptophan show overlapping peaks at about 1618, 1585, and 1609 cm\({}^{-1}\), respectively [49; 50]. The 1615-1618 cm\({}^{-1}\) peaks observed in FTIR spectra of the proteins, which are assigned to intermolecular \(\beta\)-sheet, is due to the tyrosine aromatic side chains in the FT-Raman spectra. The bands at about 1605 and 1616 cm\({}^{-1}\) were present in all reference protein FT-Raman spectra (Figure 5) and correspond to the ring vibrations from aromatic residues.