Mass Spectrometry-Based Protein Footprinting: High-Throughput MALDI Platform and Large Protein Complexes of BAF

Human’s advances in understanding biological processes rely heavily on the breakthroughs in biophysical tools. Mass spectrometry (MS)-based protein footprinting, which interrogates protein structures by measuring protein solvent assessable surface area (SASA), has grown rapidly in the last decade, successful in providing valuable data for numerous protein systems. This thesis focuses mainly on this technology.We set out to push the boundary of MS-based protein footprinting further into the new areas, preparing it for potential future applications including large-scale experiments that require high-throughput analysis the structure of large, complicated protein complexes. This thesis devotes five chapters to the method development of MS-based protein footprinting, biological questions, and the new improvements can help answer: Chapter 1 reviews the history of human’s efforts to characterize the structure of proteins, with an emphasis on the mass spectrometry-based protein footprinting. Some fundamentals are also covered in this chapter. Chapter 2 reports a method development for high-throughput analysis of protein footprinting experiments based on the MALDI ionization. By coupling the MALDI to either bottom-up peptide mapping or top-down MALDI in-source decay (ISD) strategy, we discovered the analysis time for analyzing the protein footprinting samples can be significantly shortened. In the bottom-up strategy, “coarse-grained” modification information can be reliably obtained, with sensitivity improved by employing a LC-robot conditions at a cost of analysis speed. In a top-down strategy, small footprinted samples can be analyzed rapidly. We determined the optimum conditions for conducting MALDI ISD analysis of footprinting samples and succeeded in obtaining the SASA of ubiquitin at a resolution as high as single residue level. Chapter 3 introduces a large protein complex, BRG1/BRM-associated factor (BAF). The protein attracts intensive interest for its association with human cancer, but it also raises challenges to the existing biophysical tools owing to its large size and complexity. The current knowledge of its structure, biophysical roles, assembly pathways, as well as how its structure can be probed by protein footprinting are discussed in this chapter. Chapter 4 presents the work using three footprinting methods, GEE, FPOP, and DEPC labeling to probe the structure of BAF. By carefully optimizing the LC-MS/MS instrumentation and using complementary labeling approaches, record-high amounts of modifications were identified and quantified. The footprinting data strongly support the current partial model of BAF, identify multiple conformational changes upon nucleosome binding, and reveal the dynamics of the complex. Lastly, this work exemplifies how protein footprinting can be applied to large, complicated protein systems by wisely choosing multiple modification techniques and resorting to excellent LC-MS/MS instrumentation that are tuned for this application. Chapter 5 summarizes the work in this thesis and provides a conclusion for the dissertation. We also examine the prospects for the protein footprinting approach. We believe that its role will grow in structural biology in the future