{"doi":"10.7302/6175","title":"Combining Native Ion Mobility-Mass Spectrometry and Radical-based Fragmentation for Top-down Structural Proteomics","abstract":"Proteins execute a plethora of critical cellular tasks primarily acting as multiprotein complexes, formed from selected protein isoforms, or proteoforms. Mass spectrometry (MS)-based proteomics has been at the forefront of protein characterization over the last three decades. In conventional bottom-up workflows, the peptides produced through enzymatic protein digestion are analyzed intact by MS, and then fragmented for tandem MS (MS/MS) sequencing. From the sequenced peptides, the protein components of complex mixtures can be identified. However, digestion eliminates any direct measurement of protein tertiary structure and its relationship to function. Native top-down (nTD) proteomics avoids digestion and sequences intact protein complexes under native-like conditions. Such nTD methods have proven to be effective at identifying intact protein complexes and characterizing their proteoforms; however, accessing protein sequence and 3D structure information within nTD workflows remains challenging. The first goal of this thesis is to increase the fragmentation efficiency of protein complexes in the gas-phase to enable deeper sequence coverage in nTD. In Chapter 2, protein complexes are covalently labeled with a reagent which produces charge-remote and radical-driven fragmentation under typical collision induced dissociation (CID) conditions, resulting in as much a 50% increase in protein complex sequence coverage. In Chapter 5, we explore the ability of cyclic ion mobility-mass spectrometry (cIM-MS) to achieve improved sequence coverage values in nTD experiments. Due to its improved MS and IM resolutions, we were able to observe 50% more sequence coverage obtained for protein complex precursor ions when compared with standard IM-MS tools. The second goal of this thesis is to increase the information content of structural measurements obtained in nTD experiments. Combining MS with ion mobility (IM) has allowed for the structural characterization of protein complexes by measuring collision cross section (CCS) values. Unfortunately, current IM resolution cannot distinguish proteins with small structural differences. However, collision induced unfolding (CIU) can differentiate subtly different structural proteoforms, but lacks rapid and uniform methods of data annotation In Chapter 6, CIU data acquired with cIM-MS was used to characterize the thermal stability of the NOTCH3 fusion proteins involved in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Each NOTCH3 mutant presented altered disulfide bond (determined by bottom-up proteomics) patterns, which generated different CIU pathways and stabilities. The exact order NOTCH3 unfolded was only determined by tracking the ejection a NOTCH3-specific ligand throughout the CIU pathway. In Chapter 3, CIU and electron capture dissociation (ECD) are combined to elucidate the unfolding pathways of human serum albumin dimers, enabling the complete assignment of a protein complex CIU fingerprint for the first time. In Chapter 4, a refined CIU-ECD method was developed capable assigning unfolding pathways for both 16kDa Calmodulin and 148kDa monoclonal antibody monomers. Overall, the work presented in this thesis focuses on the development of methods to increase sequence coverage and structure information for nTD workflows. Ultimately, such improved methods will lead to an enhanced understanding of protein biochemistry, human disease, as well as improved medical treatments and engineered proteins.","journal":"Deep Blue (University of Michigan)","year":2022,"id":314536,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9567,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":919422,"name":"Carolina Rojas Ramírez","orcid":"0000-0002-2085-0577","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T00:33:52.047924Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}