Reversible protein acetylation is a widespread, regulatory modification that was first discovered on histone proteins, but since has been described throughout the cell in major subcellular compartments. Several functional roles of lysine acetylation have been described including regulating enzymatic activity, protein-protein interactions, protein-DNA interactions, protein stability, and subcellular localization. With the improvements in mass spectrometry technologies, the number of identified acetylated sites has grown exponentially creating a new challenge to determine which sites are functional versus spurious. Furthermore, studies investigating the mechanisms regulating protein acetylation have primarily focused on histone acetylation, but how they may regulate non-histone protein acetylation has not been fully understood. Of importance, one of the main mechanisms through which acetylation may be regulated is through changes in the availability of acetyl-CoA. Since most acetyltransferases, including p300/CBP, have a Km near the estimated cellular concentrations of acetyl-CoA, manipulating concentrations of the substrate through activation of its production can have major effects on the levels of acetylation. Several studies have investigated the effect of knockdowns of acetyl-CoA generating enzymes (such as ACLY or ACSS2), however, few studies have measured the effects on non-histone protein acetylation. My research uses two main approaches to respond to these challenges in the field: 1) a robust, mass spectrometry method to quantify acetylation stoichiometry across the proteome, and 2) investigating dynamics of acetylation in response to changes in acetyl-CoA availability. Understanding both stoichiometry and dynamic responses during cellular stimulation are key features of interrogating the role of protein acetylation at a site- and protein-specific level. My research aims to understand the cellular mechanisms that are controlling dynamic acetylation in response to metabolic changes in acetyl Coenzyme A (acetyl-CoA) availability and production.Here, I examine the dynamics of global protein acetylation and the mechanisms regulating growth-factor stimulated dynamics, with a particular focus on how metabolism and the production and availability of acetyl-CoA is a major regulator of protein acetylation. Chapter 1 introduces protein acetylation and discusses the field's current understanding of acetylation functions, the quantitative techniques for investigating global non-histone protein acetylation, and the mechanisms involved in regulating protein acetylation. Chapter 2 presents an updated method for quantifying lysine acetylation stoichiometry and applies this method to identify serum-stimulated protein acetylation dynamics. Chapter 3 presents an in-progress project that examines the key regulatory nodes controlling growth-factor stimulated acetylation, including the kinase AKT, acetyl-CoA production through ACLY, and citrate export by SLC25A1. Chapter 4 presents the ongoing experiments and future directions for these projects. My thesis work has also allowed me to be a part of some critical collaborations that have investigated the connection between metabolism, acetyl-CoA availability, and protein acetylation in several interesting mouse models. My contributions and our key findings are presented in the Appendixes of this thesis. Appendix 1 investigates the effect of the dysregulation of the ER-localized acetyl-CoA transporter, AT-1, on protein acetylation. Appendix 2 investigates the effect of increased flux of citrate into the cytoplasm and nucleus through overexpression models of the extracellular membrane citrate transporter, SLC13A5, and the mitochondrial citrate transporter, SLC25A1, and their effect on protein acetylation. Appendix 3 investigates the effect of age, diet, and the loss of the mitochondrial localized deacetylase, SIRT3, on mitochondrial protein acetylation. Lastly, Appendix 4 examines the effects of the loss of individual ER-localized acetyltransferases, ATase1 and ATase2, on secretory pathway protein acetylation.