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Nutrient-dependent post-translational modifications (PTMs) bridge animal circadian clock and metabolism. Previous studies showed that daily cycling of protein OGlcNAcylation, a sugar-derived PTM, is driven by circadian clocks and can feedback to reinforce clock functions and daily biological rhythms. Like O-GlcNAcylation, Spalmitoylation is also a dynamic and nutrient-dependent PTM. High fat diet and palmitate treatment that may potentially affect protein S-palmitoylation are known to impact the functions of circadian clocks. I therefore hypothesize that S-palmitoylation is also a nutrient-dependent PTM underlying the interplay between metabolism and circadian clocks to regulate daily biological rhythms. Using Drosophila as an animal model, I identified many S-palmitoylation regulators with clock-controlled expression at the transcript level, suggesting that S-palmitoylation of some cellular proteins may be a clockmediated process. Among them, CG1407 that codes for a palmitoyltransferase exhibits 24-hour cycles robustly. I knocked down CG1407 genetically using RNA interference and observed the slowing down of the circadian clock and impact on daily activity rhythm. Overall, my results suggest that protein S-palmitoylation could be important for regulating daily biological rhythms. My results also highlight the utility of manipulating CG1407 expression as an experimental tool to study the interplay between S-palmitoylation cycling and biological rhythms.

Proteins perform many biological functions and characterizing their role in the cellular environment is critical in understanding a protein’s role in disease. ‘Bottom-up’ liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers the most effective strategy for large-scale global analysis of highly complex protein mixtures, i.e., proteomics. However, LC-MS/MS workflow strategies must be carefully adapted to maintain experimental reproducibility and overcome inherent limitations in detecting low abundance proteins in concentrated mixtures and labile posttranslational modifications (PTMs). This dissertation presents LC-MS/MS based analytical strategies for the direct detection and site-specific profiling of S-palmitoylated peptides, and for characterizing low abundance host cell proteins (HCPs) in purified biopharmaceutical drug substances (DS). Chapter 1 presents an introduction to quantitative LC-MS/MS-based protein analysis, including instrumentation and techniques relevant to the work presented in Chapters 2 and 3. Chapter 2 discusses S-palmitoylation, the reversible and dynamic modification of a cysteine sulfhydryl with a 16-carbon fatty acid. The dynamic cycling between a protein’s palmitoylated and depalmitoylated states regulates several intracellular events such as protein activity, localization, and protein-protein interactions. N-Ras, a well-known driver of many cancers, is S-palmitoylated near its C-terminus, and despite the therapeutic potential of targeting this modification, direct detection of this modified peptide by LC-MS/MS has not previously been successful. In fact, robust methods for direct LC-MS/MS-based analysis of S-palmitoylated proteins have been lacking. We addressed this issue by developing an LC-MS/MS “bottom-up” workflow that mitigates sample processing issues associated with S-palmitoylation, including peptide solubility, stability, LC column retention, and MS/MS-based sequence analysis. We successfully applied this workflow to directly detect and annotate endogenous acylation sites on recombinant N-Ras. Chapter 3 discusses the adaptation of an LC-MS/MS workflow for HCP characterization in biopharmaceutical drug substances. HCPs are native proteins derived from a host organism used to express biotherapeutic proteins and may be co-purified with a drug substance as process related impurities. The presence of these impurities in drug formulations may present problems in product performance and affect the health of patients. Therefore, effective monitoring of HCP levels in drug substances is essential. Common procedures for quantifying HCP content are limited to immunocapture detection methods, but serious efforts are underway in developing LC-MS/MS strategies for characterizing HCPs. Problematically, HCPs can be 106-fold less concentrated than the biotherapeutic protein in downstream products presenting a dynamic range challenge for electrospray ionization-MS. We developed a generalized, semi-automated, and plate-based hydrophilic interaction liquid chromatography (HILIC) fractionation workflow to deplete biotherapeutic protein from DS with limited sample-specific optimization. Our method is practical, quantitative, and demonstrates industry leading HCP detection sensitivity by LC-MS/MS. Chapter 4 provides a concluding summary of the novel contributions of our work and the broader impact on the field of LC-MS/MS based protein analysis. The developed method for direct detection of palmitoylated peptides promises to expand analytical profiling capabilities of an under-represented PTM. Furthermore, the demonstrated robustness and dynamic range improvements for HCP analysis show promise for assisting downstream process development of biotherapeutic drugs as part of an MS-based analytical platform.

Post-translational modifications (PTMs) play a crucial role in trafficking proteins for many location-dependent cellular functions. Protein S-acylation describes the addition of long chain fatty acids (predominantly the C16:0 palmitate) to cysteines via a thioester bond. This reversible modification thus allows for controlled regulation of protein membrane tethering during various cellular processes. Indeed, S-palmitoylated proteins include kinases, small GTPases and transmembrane receptors, which function in response to diverse signaling events. It is important to study the enzymes that catalyze this modification since it is involved in many essential pathways. Enzymes such as APT1 and APT2 have thoroughly been studied and shown to depalmitoylate a myriad of S-palmitoylated substrates and the development of selective inhibitors has accelerated the discovery of new substrates. ABHD17 has more recently been shown to depalmitoylate substrates in cell-based studies. However, not much is known about its substrate recognition mechanism and cellular function and my dissertation helps to bridge this gap in knowledge. The first chapter presents a detailed introduction to protein depalmitoylases, their different cellular roles and methods that have been developed to study them. The following chapter focuses on understanding the in vitro activity of ABHD17 and the development of potential inhibitors to study its function. The third chapter outlines a proteomics strategy that can be used to study S-palmitoylation of multiple proteins at a time and was applied to understand how ABHD17 regulates S-palmitoylation in cells. Finally, future experiments are proposed in Chapter 4 to further improve our understanding of how ABHD17 works and what its cellular function is.