UCLA

Sample complexity continues to hinder the effectiveness of Top-Down mass spectrometry, which aims to become a high-throughput platform for proteomics. One possible solution to this issue is the separation and measurement of protein mixtures using virtual 2D gel electrophoresis/mass spectrometry (virtual 2D gel/MS), where intact proteins are initially separated by isoelectric focusing on immobilized pH gradient (IPG) gels followed by mass analysis using matrix-assisted laser desorption/ionization (MALDI) MS. Here, we report on improvements made to the virtual 2D gel/MS platform. With increased automation, we have reduced the time required to acquire and visualize proteins separated on a 180 mm IPG gel from several days to under 1 hour. This automation includes the implementation of a high-speed MALDI time-of-flight mass spectrometer operating with specialized MS imaging software to acquire data. Analysis of the MS data was also automated through the development of a custom program written in MATLAB. Mass spectrometry signal intensity, signal-to-noise ratio, and sensitivity were all improved with a novel MALDI matrix application method where gels are immersed in matrix solution overnight, improving matrix crystallization. We also demonstrate for the first time the use of a 15 Tesla Fourier transform-ion cyclotron resonance mass spectrometer equipped with a MALDI source to acquire virtual 2D gel/MS data offering both an increase in resolution and accuracy of mass measurement results. Using the improved virtual 2D gel/MS technique, we identify changes to the E. coli proteome caused by both cold shock and antibiotic induced stress. The aggregation and accumulation of α-synuclein in the brain have been linked to numerous neurodegenerative disorders including Parkinson’s disease. To prevent these synucleinopathies much effort has been made to understand the cause of this protein aggregation and to find ways to prevent it. It has been shown that various ligands affect α-synuclein’s propensity towards aggregation with the small molecule compound, CLR01, a lysine molecular tweezer, decreasing aggregation and divalent heavy metals increasing aggregation. Here, we use electrospray ionization-MS and collision induced unfolding (CIU) coupled with ion mobility spectrometry to probe the effects that CLR01, Mn(II), Co(II), and Cu(II) have on the structural stability of α-synuclein in the gas phase. Our results indicate that the binding of CLR01, Mn(II), 1x Cu(II), and 3x Cu(II) all have a stabilizing effect on the structure of the protein while Co(II) destabilizes the protein. The work presented in this thesis demonstrate new mass spectrometry-based experimental platforms to qualitatively and quantitatively profile complex protein mixtures rapidly and accurately, and to probe the structural stability of protein/ligand complexes that are complementary to other biophysical methods.