# Peak Fitting Applied to Fourier Transform Infrared and Raman Spectroscopic Analysis of Proteins
## 3 Results  
### Secondary Structure Estimation for Standard Proteins  
The infrared spectra of four native proteins (concanavalin A, immunoglobulin G, lysozyme, and trypsin) were investigated. Samples were allowed to stir for an hour at room temperature before recording the spectra in order to dissolve and hydrate completely. The FTIR spectrum of pure water was subtracted from the spectra of the protein solutions. Figure 1a shows the FTIR spectrum of the standard protein solutions. All proteins show a similar peak around 1636 cm\({}^{-1}\), which does not seem to give specific information about their secondary structure. However, after subtracting the reference water spectrum from the protein spectra (Figure 1b), a more distinct spectrum with individual peaks appeared for each protein. The individual underlying secondary structure components cannot be readily observed in the amide I band (Figure 1). This is because the width of the different component bands is larger than the separation between the individual component bands peaks [37]. Thus, the second derivative of the spectrum was used as a band narrowing/peak sharpening method to identify hidden peaks. By performing peak fitting on water-subtracted spectra according to the analysis described in the Materials and Methods section, the overlapping hidden peaks were identified and reconstructed. The overall curve fitted and the second derivative of the concanavalin A spectrum are shown in Figure 2. The spectrum exhibits several discrete peaks in the amide I region. The minimum of the peaks in the second derivative spectrum corresponds to the maximum of the hidden absorbance peaks on the water-subtracted spectrum. The curve fitted and second derivative spectra of the other standard proteins are included in the Figures S1-1-S1-16). The curve fitted plots of three replications of each standard protein illustrated the high reproducibility between the replicates (Figures S1-1-S1-16). Figure 1: FTIR spectra of the standard proteins in aqueous solution (5.0% _w/v_); (**a**) original spectra (replicates), (**b**) water-subtracted spectra (replicates). The characteristic mean absorption bands of the secondary structures in proteins are indicated in Table 1. The secondary structures were estimated from (the sum of) the relative area of the peaks centered at these absorption frequencies [5, 6]. The relative amount of each secondary structure of the proteins (Table 2) is calculated from the area under the (hidden) peaks in the original spectra assigned to specific secondary structures. The component centered between 1650-1659 cm\({}^{-1}\) has been assigned to the \(\alpha\)-helix secondary structure. Bands in the region of 1610-1642 cm\({}^{-1}\) are usually assigned to \(\beta\)-sheet structure [5, 19]. The random coil conformation shows IR bands around 1643 and 1649 cm\({}^{-1}\)[19]. Upon peak fitting, four hidden bands between 1610-1640 cm\({}^{-1}\) appeared in the infrared spectrum of concanavalin A and immunoglobulin A which were all assigned to \(\beta\)-sheet secondary structures [5]. The water-subtracted spectra of concanavalin A and immunoglobulin G (Figure 1b) exhibited a very strong amide I band maxima near 1636 cm\({}^{-1}\) reflecting their high \(\beta\)-sheet content confirmed by X-ray studies [40, 41]. The peak at about 1636 cm\({}^{-1}\) was the major hidden band observed in immunoglobulin G and concanavalin A spectra, corresponding to 54% and 29% of the total amide I band area, respectively. Trypsin also showed a dominant band at 1636 cm\({}^{-1}\) comprising 70% of the total \(\beta\)-sheet content of the secondary structure. It also had \(29\pm 2\%\)\(\beta\)-turn structure based on the bands in 1660-1699 cm\({}^{-1}\) region. The water-subtracted