*3.1. Amide I and Amide III Bands Reveal Protein Secondary Structures Associated with Amyloidosis*

To investigate features of amyloid fibrils, Raman spectra of glomeruli within kidney tissues were obtained (Figure 3). Particularly, we observed peaks within amide I (1600–1700 cm−1) and amide III (1200–1300 cm−1) bands of protein, which are closely related to peptide backbone conformations, the main determinant of protein stability [11,21]. At amide I region, we observed a peak at 1658 cm−<sup>1</sup> with AA slightly shifted to a higher (1664 cm−1) frequency while AL slightly shifted to a lower (1653 cm−1) frequency, compared to the control case. In amide III spectral region, marked changes in peaks at 1239 and 1278 cm−<sup>1</sup> were observed, as peaks in AA became more distinguished whereas those in AL appeared more obscure than the NA tissue signal. Such differences are associated with secondary protein structures, particularly β-sheet and α-helix structures, which constitute amyloid fibrils [10,21,29]. The AL spectrum exhibits peaks at 1306 and 1334 cm−1, attributed to sidechain vibrations [11]. In addition, we observed subtle peaks in a higher wavenumber region, associated with changes in lipids. Peaks around 1552 and 1582 cm−<sup>1</sup> represent aromatic amino acids, such as tryptophan and phenylalanine [21]. The intensities in the observed bands, 1582 cm−<sup>1</sup> of AL tissue, and 1658 cm−<sup>1</sup> of AA tissue, vary due to the non-uniform distribution of the amyloid deposits, as marked by the heterogeneity of amyloid-positive samples. In addition, the polymorphism of fibrils may augment the heterogeneity [5]. To assess the changes in protein structures arising from amyloid fibrils, Raman band areas of amide I, amide III, and phenylalanine were evaluated (Figure 3b–d). The amide I band area of AL (Figure 3b) appeared evidently higher than the others, whereas the amide III band area of AA (Figure 3c) showed a clear distinction from the others. In addition, an increase in phenylalanine band area is observed in the AL spectra (Figure 3d), with a statistically significant difference from the band area under the AA or NA tissue spectra. Such an observation indicated that both AA and AL fibrils consist of protein secondary structures with varying contributions of C-N stretching, N-H bending, and C=O stretching vibrations [21].

To further investigate the influence of amyloid fibrils depending on the associated tissue site, we expanded the examination of the Raman spectra of glomeruli, marked in Figure 4a, as well as outside of the glomerulus region. Figure 4b shows distinct spectral profiles for each amyloid type at both glomerular and non-glomerular sites. The corresponding second derivative analysis is shown in Figure 4c. We performed second derivative analysis to objectively identify sharp changes in spectra and locate their vibrational bands, enabling us to further distinguish characteristic spectral features [9,11,44]. Second derivative analysis of amide I, II, and III bands revealed spectral components and peak shifts unnoticed in Raman tissue spectra. Analysis of AA glomerular regions exhibited a split in the 1213 cm−<sup>1</sup> band, with prominent peaks around 1265, 1305, and 1584 cm<sup>−</sup>1, associated with the mixture of β-sheet and α-helix structures. The contributions of protein secondary structures in AL fibrils were different from those in AA fibrils, with peaks observed around higher Raman bands, at 1625, 1641, and 1655 cm<sup>−</sup>1, mainly attributed to C=O stretching vibration. These observations are consistent with previous reports that indicate both AA amyloidosis and AL amyloidosis exhibit protein secondary structures, as the misfolded AA and AL proteins, respectively, aggregate, form amyloid fibrils, and adopt a β-sheet conformation [45]. Second derivative analyses reveal that Raman spectroscopy can molecularly distinguish this common structural feature (β-sheet) across AA and AL amyloidosis, as shown by their distinct Raman bands.

**Figure 3.** Raman spectroscopy of frozen kidney tissue featuring amyloid deposits. (**A**) Raman spectra of glomeruli within AA, AL, and NA tissues. Each spectrum represents an averaged and normalized spectrum with 1 standard deviation shaded. They are normalized on the spectral region assigned to water (3100–3400 cm−1), assuming an equivalent water content for all samples. Raman band area analyses of (**B**) amide I (1600–1700 cm−1), (**C**) amide III (1200–1300 cm−1), and (**D**) phenylalanine (1582 <sup>±</sup> 3 cm<sup>−</sup>1) of AA, AL, and NA glomeruli. Statistical significance: \*\*\* *<sup>p</sup>* < 0.0001.

**Figure 4.** *Cont.*

**Figure 4.** (**A**) Microscopic image of frozen kidney tissue identified with the glomerulus. Scale bar = 100 μm. (**B**) Averaged and normalized Raman spectra collected within and without glomeruli of AA, AL, and NA tissues with 1 standard deviation shaded. (**C**) Second derivative analysis of phenylalanine (1000–1500 cm<sup>−</sup>1), amide III (1200–1350 cm−1), and amide II-I (1550–1700 cm−1). Each spectrum in (**B**,**C**) is color-coded based on the type and deposition site and plotted in order, from top to bottom: AA-within glomeruli, AA-without glomeruli, AL-within glomeruli, AL-without glomeruli, and NA.
