UC Riverside

Comprehending protein structures is fundamental to understanding their functions and interactions at the molecular level. Long-lived human proteins (LLPs) are susceptible to damage associated with aging-related diseases such as cataracts and Alzheimer's disease (AD). Alternation in chirality from L to D in canonical amino acids does not have any mass shift, yet, it can modify the protein structure, alter its functions, and disrupt the cellular balance. Analytical techniques such as liquid chromatography and mass spectrometry (MS) have been utilized to identify isomers in LLPs. MS is a powerful and versatile tool for proteomic studies and structural characterization. Despite peptide isomers having no mass shifts, MS can differentiate isomers through their fragmentation patterns. This dissertation presents several innovative MS-based strategies for identifying and locating peptide isomerized residues. Our statistical framework can distinguish several types of isomers, including L/D stereoisomers and Leu/Ile, even with some standard fragmentation methods, such as collision-induced dissociation (CID) and higher-energy dissociation (HCD). This method can identify peptide isomers collected via direct infusion and liquid chromatography LC-MS. We successfully identified various isomers in human eye lens lysis, which shows the potential application to other biological samples. Secondly, incorporating ion mobility spectrometry (IMS) with mass spectrometry has also been utilized for isomer differentiation. The isomeric fragment ions were separated in the gas phase and had different arrival times based on their structural differences. This arrival time shift can locate in the isomerized residues, while the fragmentation patterns can also be used to identify isomers. This method provides two-dimensional data that enables confident isomer differentiation. Lastly, an efficient and accessible method for locating isomerized residues has been introduced using tandem mass spectrometry, which includes an improved R-value method incorporated with statistical analyses. The isomerized site can be pinpointed in peptides using CID-CID or CID-HCD. This method is accessible for instruments equipped with ion traps and allows for trivial isomer identification. The efficient and accessible method allows for the quantification of isomers in a mixture, and identifying isomerized residue in LLPs may reveal the relationship between isomerization and aging diseases.