Studies on brain aging and Alzheimer's disease using mass spectrometry, organic synthesis, and biostatistics
Keywords: Chemistry.

Brain aging is a poorly understood process widely understood to be a major causative factor in age-associated neurodegenerative diseases such as Alzheimer's disease (AD). Proteins are involved in brain aging and neurodegeneration in many ways, and their functions and dysfunctions are dependent upon their abundances and their three-dimensional chemical structures. Emergent tools in multiple chemistry subfields, including mass spectrometry-based proteomics, enable a more detailed understanding of the states of proteins in these systems. In this Dissertation, I describe the usage of two chemistry techniques, primarily mass spectrometry-based proteomics and secondarily synthetic organic chemistry, to study proteins involved in brain aging and AD. In addition, I describe the utilization of existing statistical methods and the development of new statistical tools to further enhance our understanding of changing proteomes. The first Chapter in this Dissertation contextualizes my work in the Wyss-Coray Laboratory by providing the background for my transition from the Department of Chemistry at Stanford University, where I earned a Master of Science degree, to the Department of Neurology and Neurological Sciences in the Stanford University School of Medicine. This Chapter lays the groundwork for the lens, rooted in chemistry, through which I have viewed the fields of brain aging and AD as I have pursued subsequent studies in the Wyss-Coray Laboratory. Chapters 2 and 3 serve as technical introductions. Chapter 2 presents historical and biological background on the fields of brain aging and AD with an emphasis on the role of proteins. In Chapter 3, mass spectrometry-based proteomics is introduced, first with an in-depth conceptual and practical review of basic aspects, and then with an introduction to two advanced topics relevant to my work in later chapters: data-independent acquisition (DIA) and limited proteolysis-mass spectrometry (LiP-MS). The primary results presented in this Dissertation are the subjects of Chapters 4--7. In Chapter 4, the centerpiece of the Dissertation, the main focus of my graduate work is presented. This Chapter describes the usage of LiP-MS to study brain aging by assessing changes in both protein abundance and protein chemical structure that occur in the cerebrospinal fluid (CSF) with aging. It describes modifications of preexisting LiP-MS analysis methods that I employed to address challenges posed by the study. The resulting analytical workflow was developed and packaged into a tool that will allow others to easily perform sophisticated LiP-MS analyses to study mammalian biology. This work culminated in the discovery of a covalent immunocomplex in CSF previously unknown to exist in CSF, the Cd5l-IgM complex, whose abundance increases significantly in the CSF during aging. Chapter 5 discusses one persistent weakness of LiP-MS: LiP-MS results are ambiguous with respect to specific chemical/structural change. Also, it is still not entirely known which chemical changes in proteins are detectable by LiP-MS and which are not. In particular, changes in N-glycosylation, a post-translational modification found on the vast majority of CSF proteins, has not been shown to be detectable by LiP-MS. By enzymatically removing N-glycans from CSF proteins using PNGase F and performing LiP-MS, LiP-MS was shown to detect structural changes caused by changes in N-glycosylation for the first time. This Chapter also discusses the successful usage of the asparagine deamidation modification, a byproduct of PNGase F activity, to provide further evidence of deglycosylation and explore the relationship between N-glycosylation and protein chemical structure. Surprisingly, the ensuing analysis suggested that removal of N-glycans can induce binding of ligands or metals to proteins even at a distant site. The most striking example of this phenomenon was found in transferrin, highlighting a previously unknown possible role of N-glycosylation in physiological iron uptake and/or transport. Chapter 6 describes a theoretical treatment of an important component of the statistical analysis done in Chapters 4 and 5: the correction of p-values for multiple hypothesis testing. This Chapter discusses the costs and benefits of p-value correction in quantitative omics experiments, including a model of quantitative omics that includes subtle, realistic distinctions between the null hypothesis H0 and the alternative hypothesis HA which were necessary to simulate data that resembled real-world quantitative omics. Here I identify permutation-mediated false discovery rate (FDR) estimation as an effective and preferable tool to Benjamini-Hochberg method of p-value correction in most cases, and exceptions are discussed. I show that any true change can be detected in a well-designed proteomics experiment while adjusting p-values. Chapter 7 presents the use of a different chemistry technique, synthetic organic chemistry, to study a specific protein relevant to brain aging and AD: TGF-β. Liver metabolism of a small-molecule drug candidate, C381, has complicated studies on TGF-β, and this Chapter conclusively elucidates the structures of the hepatic metabolites of C381 via chemical synthesis. Liver enzymes selectively hydroxylate C381 at a cyclohexyl substituent in the presence of weaker C--H bonds and more electron-rich carbons. The resulting analogs were tested for their potency in activating TGF-β, identifying the parent compound, C381, as the active species. The synthetic studies also reveal surprising changes in the chemical properties of intermediates upon the addition of a single oxygen atom to the structures, which can greatly complicate synthesis even with the sensible involvement of protecting groups. Together, these Chapters present some of the surprising, illuminating, and relevant discoveries that can be made in the fields of brain aging and AD when chemistry and statistics are employed