Development of Raman detection methods for proteomic applications
The purpose of this research is to develop useful Raman scattering methods for proteomic applications including protein and peptide identification as well as the detection and quantitation of post-translational modifications, structural changes and protein-ligand interactions. The first five chapters are focused on the development and applications of the drop coating deposition Raman (DCDR) method. Chapter one describes DCDR and its advantages as a general method for protein detection. Chapters two and three describe applications of DCDR to the analysis of peptide phosphorylation. Chapter two describes the strong Raman fingerprint of tyrosine-phosphorylation in peptides with different primary sequences and the use of multivariate analysis for quantifying tyrosine phosphorylation. Chapter three focuses on using pH modulation and spectrometric titration to enhance the detection of bound phosphate, and to track its protonation equilibrium. Chapter four describes preliminary studies aimed at developing a general method for predicting the Raman spectra of peptides using a library composed of DCDR spectra of a set of trimer peptides each differing by a single amino acid residue. In chapter five, we demonstrate an ultrafiltration-Raman-difference (UFRD) method which combines ultrafiltration and DCDR. The UFRD method is demonstrated to be useful for both qualitative and quantitative analysis of the protein ligand binding constants and interactions, for applications as a tag-free drug screening. Chapter six describes a new isotope edited internal standard (IEIS) method for use as an internal standard for quantitative surface enhanced Raman scattering (SERS) and surface enhanced resonance-Raman scattering (SERRS) applications. It is shown that IEIS can be used to quantify concentrations over the pM to μM concentration range with an accuracy of a few percent. Thus, IEIS shows significant promise in overcoming the poor reproducibility and quantitation capability of colloidal SERS/SERRS to produce a higher performance replacement for conventional fluorescence tags.
Ben-Amotz, Purdue University.
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