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Protein modification is the process by which the order of amino acid sequencing is rearranged or the functional groups on the amino acids are altered by a chemical process. Protein modification is responsible for processes such as metabolism, metastatic behavior, mental cognition and muscular movement [1]. In order to understand protein modification through amino acid alteration, it is first necessary to understand the real time behavior of the amino acid groups under stress as well as develop a method to analyze multiple amino acids within a single sample. Our goal was to analyze the resilience of the amino acids and to understand the impact their presence would have on the specificity of the Raman bands that indicate spectral fingerprint or vibrational modes. A blend of three branched chain amino acids, L-leucine, L-valine and isoleucine, were investigated for stress analysis and strain effects due to local heating of the sample through confocal Raman spectroscopy and compared to individual amino acid Raman spectroscopy. Data was collected using a full factorial design of experiment (DOE) with the factors including laser power, acquisition time, and spectral range, where each was repeated three times. With a laser spot size of approximately one-micron in diameter, the laser power and acquisition times were varied over the spectral range of 50-3600 cm-1. The crystal size of the blended samples were between 5-10 μm in width and 10-15 μm in length. The crystal size of the individual L-leucine and L-valine samples were between 20-40 μm in diameter. The individual isoleucine samples were between 15-20 μm in width and 60-80 μm in length. Assignment of each band in the blend derived from previously reported bands for the respective amino acids, where L-leucine influenced the majority of the Raman bands observed. The spectra collected from the blended sample were compared to the individual spectra collected, and when the laser irradiance was increased, local heating effects gave rise to bands stemming from the Lvaline and isoleucine indicating the relative resilience of the individual amino acids. Our samples yield high spectral response in the 2800-3100 cm-1 range owing to the C-H bonds of each component. This is expected as our samples were comprised of three-branched chain amino acids with functional groups comprised solely of C-H bonds. When analyzing a multicomponent amino acid sample where characteristics of the minor constituents are desired, Raman spectroscopy can play a significant role in revealing the underlying structure. Principal component analysis (PCA) was performed using each of the individual spectra and the blended spectra. PCA showed the first coefficient for Lleucine was responsible for approximately 15% of the blended spectra.
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