UC Riverside

The proper structure and function of proteins in living organisms is essential to life. Even small changes to the amino acid residues that make up proteins can drastically alter both structure and function. As proteins age, they accumulate post-translational modifications and spontaneous chemical modifications such as isomerization, which can drastically affect both structure and function. In long-lived proteins (LLPs) where little to no turnover occurs, significant amounts of isomerization can build up over time. Long-lived proteins have been implicated in a growing number of human health conditions. Thus, understanding the relationship between these LLPs and isomerization is growing in importance. Additionally, the connection between isomerization and protein structure/function is complex and requires further unraveling to understand completely. In this work, we detail new mass spectrometry methods for identifying isomerization, and explore the consequences of isomerization on enzymatic digestion. First, we start by examining cis/trans proline isomerization and establish that mass spectrometry can distinguish these isomers if proper care is taken to select the appropriate ESI conditions along with charge solvation to preserve solution-state structure into the gas phase. Next, we detail a L-isoAsp-specific covalent labeling technique and validate this method on a 72-year-old eye lens sample. This technique allows major isomerization sites to be quickly and easily identified through introduction of a mass shift which can be searched for just like a regular post-translational modification. Following that, the consequences of Asp isomerization in substrates of trypsin-like proteases are studied, revealing that even robust enzymes such as trypsin are unable to recognize isomerized residues in the active site. In addition, the 20S proteasome is found to be unable to digest isomerized substrate, carrying important implications in the ultimate fate of isomerized LLPs. Finally, we examine the effects of using higher-energy collisional dissociation (HCD) to excite the hydrogen-deficient radical formed during radical-directed dissociation (RDD). RDD-HCD was found to create different fragments depending on the amount of HCD energy used and revealed insight into radical-based fragmentation. Overall, this work highlights mass spectrometry as a powerful tool to study the structural and functional consequences of isomerization in proteins.