UC Riverside

Photodissociation (PD) mass spectrometry has been shown to be a useful analytical technique for determining protein modifications. Described herein is the development and application of photodissociation for various modifications. Photo-excitation with 266nm laser light normally does not yield fragmentation. However C-S, S-S, C-I, C-Br, and C-Cl bonds can be homolytic cleaved through direct dissociation pathways. This selective fragmentation can be utilized to identify peptides or proteins of interest. Radicals generated through this process can cause further dissociation of the peptide backbone. Radical directed dissociation (RDD) is useful for identifying the location of modification sites. The selective nature of fragmentation is shown to provide facile identification of PTM sites by greatly simplifying data analysis. Reported herein is the discovery of a novel gas phase dissociation technique useful for the facile analysis of protein modifications.

Phosphorylation sites are selectively modified through â elimination and Michael addition chemistry, installing photolabile group. Photodissociation yields homolytic cleavage of the C-S bond at the modification site, generating a â radical which is poised to fragment the peptide backbone selectively at the previously phosphorylated residue, thus allowing facile identification. Cysteine residues, uniquely reactive amino acids, are shown to form covalent bonds with quinones. Furthermore, the chromophoric properties can be leveraged for site specific photodissociation. Photodissociation reveals both the presence and location of modified cysteine residues. Selective fragmentation of a single bond in a whole protein is demonstrated. PD can be used to determine both the presence and site of modification generated by naturally occurring molecules, such as dopamine, which can harness quinone chemistry to modify proteins.

Also reported is the discovery of photodissociation at 266 nm to selectively cleave disulfide bonds in the gas phase, while leaving all other bonds intact. This methodology can be used to identify disulfide bonded pairs in complex systems. LC-MS experiments utilizing photodissociation were developed for analysis of protein digests. Peptides containing biomarkers of oxidative stress can be easily identified by LC-PD-MS. Proteins exposed to oxidative stress can be halogenated at tyrosine residues. Homolytic cleavage of the carbon-halogen bond is a favorable process and allows facile identification of these types of biomarkers. Shown in this dissertation is the selectivity offered by photodissociation; homolytic cleavage allows simplification of data analysis by quickly identifying the peptides of interest in a mixture while subsequent selective backbone fragmentation allows facile analysis of protein modifications.