Book Description

Biological information flows as DNA is transcribed into mRNA and then translated into proteins. However, sequence variations from mutations and alternative splicing events combined with post-translational modification (PTMs) of proteins result in a diversity of protein forms (referred to as proteoforms) that can arise from a single gene. Mass spectrometry (MS)-based proteomics provides an unprecedented opportunity to understand the role of proteoforms in health and disease; however, many challenges remain. For example, despite their importance as drug targets (>50% of current drugs), membrane proteins are traditionally underrepresented using MS-based proteomics because of their lower expression level, hydrophobicity, and lack of established protocols. To address these challenges, I developed a novel photocleavable surfactant, Azo, which can effectively solubilize proteins and is compatible with MS analysis (Chapter 2). We demonstrated Azo-aided top-down proteomics (the analysis of intact proteins by MS) enabled the solubilization of important membrane proteins from biological samples, including heart tissues, for comprehensive characterization of their proteoforms. Moreover, Azo is simple to synthesize and can be used as a surfactant in polyacrylamide gel electrophoresis. We next incorporated the surfactant technology to facilitate high-throughput bottom-up proteomics (the analysis of digested proteins by MS) for more extensive proteome coverage and protein expression quantification. Furthermore, we established simple, high-throughput membrane and extracellular matrix proteomic methods using Azo (Chapter 3-4). Combining Azo-aided bottom-up and top-down proteomics, we established a powerful integrated strategy to extensively characterize proteins from biological samples. Finally, a novel membrane protein enrichment and multidimensional liquid chromatography separation strategy was developed to further expand the scope of MS-based top-down proteomics for characterizing the membrane proteoform landscape (Chapter 4). Future development and applications of MS-based approaches for the characterization of membrane proteoforms are discussed in Chapter 5.