UCLA

Mass spectrometry (MS) has been found to be a useful technique for the study of various compounds. Fundamentally, MS is a way to measure the masses of compounds for the purpose of identifying and quantifying those compounds. Protein mass spectrometry, where proteins are the analyte of interest, has been found to return relevant information on those proteins including the mass, the identity and location of modifications, and even aspects of protein higher-order structure. The work here aims to develop mass spectrometry methods for the study of proteins and to use those methods to investigate amyloid proteins common in neurodegenerative diseases. Herein, it is described how ClipsMS, a program that assigns internal fragments resulting from top-down mass spectrometry (TD-MS), can be utilized to increase the sequence coverage of proteins and locate modifications. This work also illustrates how native TD-MS of large protein complexes with high-energy C-trap dissociation (HCD) can release covalent fragments that reveal aspects of higher-order structure. Furthermore, it is described how TD-MS of proteins with electron capture dissociation (ECD) on an orbitrap-based mass spectrometer can return relevant information on proteins including sequence information, the location of modifications, and higher-order structure information on protein complexes. In this work, MS techniques were also utilized to characterize neurodegenerative disease protein monomers and oligomers. The research conducted reveals the location of phosphorylation sites on commonly phosphorylated amyloid proteins and that phosphorylation compacts the gas-phase structure of those proteins. It is possible that structure alteration due to phosphorylation could modulate the aggregation potential of amyloid proteins. In addition, it was found that CLR01, a molecular tweezer compound that has been found to inhibit amyloid protein aggregation, directly interacts with the N-terminus of multiple proteoforms of the amyloid protein α-synuclein and that the molecule compacts the gas-phase structure of the protein. It is possible that compaction of the N-terminal region of α-synuclein by CLR01 could prevent monomers from interacting with one another and forming oligomers and fibrils of the protein. Lastly, MS was utilized to determine size information and aggregation interface information for various amyloid protein oligomers. Characterization of these small aggregates could provide information on how they become toxic in brain neurons. The data presented here aims to further mass spectrometry methods for the characterization of proteins and to use those methods to reveal protein aggregation mechanisms and discover possible therapies for neurodegenerative diseases.