Shining the Light on Aging and Isomers Using Photodissociation and Radical Chemistry

Biological aging is a complex and nuanced chemical process that proceeds along numerous routes at the molecular level. Spontaneous deamidation of asparagine and isomerization of aspartic acid are among the most prevalent age-related chemical modifications and are associated with a growing list of human diseases. Although both degradation pathways are common throughout the body, they are often unnoticed because the resulting chemical modification is relatively minor and is exceptionally difficult to detect. Structural characterization is further complicated because both deamidation and isomerization produce four isomers of aspartic acid (L-Asp, D-Asp, L-isoAsp, and D-isoAsp). To better understand the aging process, we utilized radical directed dissociation (RDD) in conjunction with mass spectrometry to identify and quantify the products of deamidation and isomerization. We began by outlining intrinsic factors that govern the deamidation rate, and external factors that influence product outcomes to develop models of peptide and protein aging. Subsequent studies revealed the specific structural and functional perturbations associated with the unnatural isomers present in aged proteins. To further expand our isomer detection capabilities, we applied our radical based fragmentation method to the glutamine deamidation, which exhibits several parallels to asparagine deamidation, but has remained largely uncharacterized. Importantly, we demonstrate that radical chemistry generated diagnostic and informative fragment ions for both glutamic acid and isoglutamic acid. We apply this technique in a manner that is amenable to shotgun proteomics and reveal several key differences between the two aging processes. Finally, we tailor our radical based analytical methodology toward the analysis of isomeric glycans. After successfully discriminating a comprehensive family of isomeric glycans with RDD, we demonstrate similar capabilities with 213 nm ultraviolet photodissociation, and outline how such a versatile approach may unify the closely related fields of glycomics and proteomics