Developing Selective-Dissociation for Proteoform Characterization in Top-Down Proteomics

Summary This project seeks to develop enabling technology for the characterization of all human proteoforms. Proteoforms are the active species in biology and include not just the genetically encoded protein sequence but any alterations including alternate splicing, post-translational modifications, spontaneous chemical modifications, etc. As such their structure cannot be readily inferred from genomic data. Identification and characterization of proteoforms is the first step towards developing a complete mechanistic understanding of the cellular processes underlying both health and disease. Proteoforms are large, complex molecules that can be difficult to disambiguate. For example, an important functional difference could result from changing the location of a single modification, creating isomers that would appear to be nearly identical in an analytical sense. However, differences in key areas would distinguish these two proteoforms, and this proposal will develop methods to target those key differences by rationally and specifically fragmenting proteoforms at desired residues inside a mass spectrometer. This approach is an improvement over existing methods where information content is dependent on random fragmentation events, which may or may not reveal the key areas that differ. To enable rational protein dissection in the gas phase, chemical modifications must first be installed to create a weak link in the backbone chain that can be specifically triggered. The proposal will develop novel photoactivated modifiers that will selectively create radicals that will facilitate selective dissociation. Both the synthetic modifications and subsequent mass spectrometry are crucial innovations that will result from this research and further advance our ability to characterize proteoforms with the end goal of populating a human proteoform atlas. Project Number: 1R01GM158899-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Ryan Julian (+1 co-PI) | Institution: UNIVERSITY OF CALIFORNIA RIVERSIDE, RIVERSIDE, CA | Award Amount: $396,886 | Activity Code: R01 | Study Section: Enabling Bioanalytical and Imaging Technologies Study Section[EBIT] View on NIH RePORTER: https://reporter.nih.gov/project-details/11294102