Tauopathies refer to a group of neurodegenerative diseases that are characterized by pathological aggregations of the microtubule-associated protein Tau (MAPT). Aberrant alternative splicing of Tau exon 10 leads to the imbalanced expression of Tau isoforms that contain either 3 or 4 microtubule binding repeats (3R-Tau or 4R-Tau) and this is sufficient to cause the formation of Tau inclusions. Nonetheless, the exact molecular mechanisms that regulate aberrant Tau exon 10 splicing regulation and subsequent Tau aggregation in tauopathies remain elusive. In my thesis research, I used RNA Antisense Purification by Mass Spectrometry (RAP-MS) to identify upstream regulators of Tau splicing events. Among the 15 identified novel protein candidates, I validated that hnRNPA2B1 and hnRNPC are required to promote 4R-Tau expression, whereas hnRNPH1 supports 3R-Tau expression. Separately, to elucidate the functional difference between 3R- and 4R-Tau isoforms, I performed proximity-dependent biotin identification (BioID2) for all six human central nervous system Tau isoforms in mouse primary neurons. Followed by tandem mass tag (TMT)-labeling proteomics and data analysis, I observed that 4R-Tau proximal proteins are highly enriched in endocytosis, whereas 3R-Tau proximal proteins show top enrichment in fatty acid metabolism. Through further biochemical validations, I found that MAT2A, a S-adenosylmethionine synthase, has higher binding affinity with 3R-Tau versus 4R-Tau. Overall, using novel proteomics methods, I discovered novel Tau splicing regulators and characterized the neuronal Tau isoform-specific proximity proteome networks. These proteins, once validated through future functional studies in cellular and animal models, can represent therapeutic and diagnostic targets for neurodegenerative tauopathies. / Thesis / Doctor of Philosophy (PhD)

In this work, tandem mass spectrometry (MS/MS)-based quantitative protocols were developed to facilitate differential protein expression analysis and biomarker discovery via a two-step sample interrogation strategy: (a) global protein profiling and differential expression analysis by spectral counting; and, (b) biomarker candidate validation by targeted screening, i.e., multiple reaction monitoring (MRM). Preliminary experiments were performed to evaluate the performance of the spectral counting method. The method proved to be applicable for proteins with spectral counts≥2, and a close-to-linear relationship between protein concentration and spectral count data was achievable at protein concentration levels <0.1 μM. The detection limit was 40-800 fmol. A protein/peptide library containing ~10,000 peptide entries that facilitates the development of future MRM experiments, was developed. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum, the peptide retention time, and the top 10 most intense product ions that correspond to a given parent peptide. Only proteins identified by at least two spectral counts are listed. An MRM experiment was performed to demonstrate the successful applicability of this peptide library for the identification of putative biomarkers in proteomic samples. / Master of Science

In vitro liver models that closely mimic the in vivo microenvironment are central for understanding hepatic functions and intercellular communication processes. Bottom-up shotgun proteomic analysis of the hepatic cells can lend insight into such processes. This technique employs liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of protein abundances by measuring intensities of their corresponding peptides. Organotypic 3D liver models have been developed in our laboratory that consist of hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM), which serves as a mimic for the Space of Disse. Each component within these models is easily separable allowing for systematic evaluation of the cells and PEMs. In this study, proteomes of hepatocytes from PEM containing models, cultured with and without LSECs, were compared to those from monolayers. Changes in core metabolism were evaluated among all culture conditions. Overall, all cultures were ketogenic and performed gluconeogenesis. The presence of the PEM led to increases in proteins associated with mitochondrial-based β-oxidation and peroxisomal proteins. The PEMs also limited production of structural proteins, which are linked to dedifferentiation of hepatocytes, suggesting that cell-ECM interactions are essential for maintenance of their liver-like state. The presence of LSECs increased levels of carboxylesterases and other phase I and phase II detoxification enzymes suggesting that intercellular signaling mediates enzyme abundance. Taken together, these results suggest that the cell-cell (from the LSECs) and cell-ECM (from the PEMs) interactions exert different, yet crucial effects, and both are required for the preservation of metabolic liver functions and differentiated phenotypes. Changes in the PEMs as a result of cell culture were also evaluated but exhibited minimal differences at this time point. Proteomes of LSECs monolayers were also characterized. Enzymes related to the metabolism of amino acids, lipids, oxidative phosphorylation and phase I and phase II detoxification processes were all identified in LSECs monolayers highlighting their role in these processes. Characterization of 3DHL LSECs was not possible due to ion suppression resulting from the presence of excess contaminant proteins. Nonetheless, this study provides a foundation in which LSECs from 3D liver models can be compared against in future studies. / Ph. D.

Thesis advisor: Eranthie Weerapana / Protein activity is modulated by hundreds of post-translational modifications (PTMs). This thesis will describe the development and application of proteomic methods to study three chemically distinct PTMs. In the first project we describe the development of a proteomics platform to identify cysteine oxidation sites on interactors of the NADPH oxidase complex in response to EGF activation. The NADPH oxidases (Nox) are the source of H(2)O(2) which acts as a secondary messenger during EGFR activation. Known targets of Nox H(2)O(2) include phosphatases PTP1B and PTEN. Oxidation of the active site of PTP1B and PTEN temporarily inactivates their phosphatase activity which allows for EGF signal propagation. The platform involves combining TurboID with OxICAT to identify proteins which are oxidized by Nox2 in a spatially and temporally controlled manner. In the second project, our goal is to identify proteins which recognize two Met oxidation sites in actin known to play a role in regulating the transition between F and G actin. We utilized a peptide based photo-crosslinking approach to identify PFKL and HSP70s HSPA8 and HSPA1B as putative "readers" of oxidized or unoxidized methionine in actin respectively. Finally, protein citrullination is a enzyme catalyzed PTM where the guanidinium on arginine is converted into a urea by a family of enzymes called protein arginine deiminases. Aberrant citrullination is linked to many human diseases including rheumatoid arthritis. Therefore, proteomic methods to characterize citrullination can provide insights into disease pathophysiology. We describe the identification of novel protein targets of with a chemoselective biotin phenyl glyoxal probe, and the development of a label free proteomic method to identify sites of citrullination. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The aim of this project was to get insight into mass spectrometry based proteomics, get familiarized with novel techniques, and obtain the operating skills with modern Orbitrap mass spectrometers. In order to achieve this, the proteome changes in neuropathic pain responses corresponding to nerve injury side in individual rat’s spinal cord were explored. We focused in protein identification and quantification of the expressed proteins in 3 different set of samples, SNL, Sham and Naive rats.

Chan Ching-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 109-123). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.vi / Abbreviations --- p.vii / Table of contents --- p.xi / List of figures --- p.xiv / List of tables --- p.xv / Chapter 1. --- Introduction --- p.1 / Chapter 2. --- Literature review --- p.3 / Chapter 2.1 --- History --- p.3 / Chapter 2.2 --- The living environment and life cycles of Cordyccps --- p.3 / Chapter 2.3 --- Chemical constituents of Cordyceps --- p.4 / Chapter 2.3.1 --- Determintaion of active ingredients in Cordyceps --- p.6 / Chapter 2.4 --- Therapeutic Functions --- p.8 / Chapter 2.4.1 --- Cardiovascular and circulatory functions --- p.8 / Chapter 2.4.1.1 --- Effects on Cholesterol and lipid metabolism --- p.8 / Chapter 2.4.1.2 --- Dilation of vasculature and cerebolature --- p.8 / Chapter 2.4.2 --- Respiratory functions --- p.9 / Chapter 2.4.3 --- Renai functions --- p.9 / Chapter 2.4.3.1 --- Effccts on chronic renal failure patients --- p.9 / Chapter 2.4.3.2 --- Protective effects on kidney toxicity --- p.9 / Chapter 2.4.4 --- Hepatic functions --- p.10 / Chapter 2.4.4.1 --- Effect on hepatitis B patients --- p.10 / Chapter 2.4.4.2 --- Energy state of liver --- p.10 / Chapter 2.4.5 --- Aging and senescence: Longevity enhancement --- p.11 / Chapter 2.4.5.1 --- Senescence --- p.11 / Chapter 2.4.5.2 --- Antioxidant effects --- p.11 / Chapter 2.4.6 --- Immune functions --- p.12 / Chapter 2.4.6.1 --- Enhancing immune system --- p.12 / Chapter 2.4.6.2 --- Anti-tumor effects --- p.12 / Chapter 2.4.7 --- Reproductive effects --- p.13 / Chapter 2.4.8 --- Hyperglycemic effects --- p.13 / Chapter 2.5 --- Cultivation --- p.15 / Chapter 2.5.1 --- Carbon and nitrogen sources --- p.15 / Chapter 2.5.2 --- Initial pH and temperature --- p.16 / Chapter 2.5.3 --- Bioelements --- p.16 / Chapter 2.5.4 --- Agitation intensity --- p.16 / Chapter 2.5.5 --- Aeration rate --- p.18 / Chapter 2.6 --- Fungal genetics --- p.19 / Chapter 2.6.1 --- EST approach --- p.19 / Chapter 2.7 --- Proteomic studies --- p.21 / Chapter 2.7.1 --- 2D gel electrophoresis --- p.21 / Chapter 2.7.2 --- Mass spectrometry --- p.22 / Chapter 2.7.3 --- Limitations and improvements --- p.22 / Chapter 2.7.4 --- Multiple spots for the same proteins --- p.24 / Chapter 2.7.5 --- Fungal proteomics --- p.24 / Chapter 2.7.5.1 --- Extraction method --- p.24 / Chapter 2.7.5.2 --- Combined uses of EST sequences and amino acid sequences --- p.26 / Chapter 2.7.5.3 --- Glycosylation --- p.26 / Chapter 3. --- Materials and methods --- p.28 / Chapter 3.1 --- Genomic Studies --- p.28 / Chapter 3.1.1 --- Strains and growth conditions --- p.28 / Chapter 3.1.2 --- Total RNA Extraction --- p.28 / Chapter 3.1.3 --- Isolation of mRNA --- p.30 / Chapter 3.1.4 --- cDNA Library Construction --- p.30 / Chapter 3.1.5 --- PCR Screening --- p.31 / Chapter 3.1.6 --- EST sequencing --- p.31 / Chapter 3.1.7 --- EST assembling and annotation --- p.32 / Chapter 3.2 --- Proteomic Studies --- p.33 / Chapter 3.2.1 --- Sample Preparation --- p.33 / Chapter 3.2.2 --- Quantitation --- p.34 / Chapter 3.2.3 --- 2-D PAGE --- p.34 / Chapter 3.2.4 --- In-gel digestion and peptide extraction --- p.36 / Chapter 3.2.5 --- MALDI-TOF MS Analysis --- p.37 / Chapter 3.3 --- Determination of adenosine using RP-HPLC --- p.38 / Chapter 4. --- Result --- p.39 / Chapter 4.1 --- Genomic studies --- p.39 / Chapter 4.1.1 --- cDNA library --- p.39 / Chapter 4.1.2 --- cDNA sequence analysis --- p.39 / Chapter 4.1.3 --- Functional annotation and analysis --- p.42 / Chapter 4.2 --- Proteomic --- p.67 / Chapter 4.2.1 --- 2D analysis and resolution --- p.67 / Chapter 4.2.2 --- Protein identification and annotation --- p.74 / Chapter 4.2.3 --- Image analysis --- p.81 / Chapter 4.3 --- Presence of adenosine (HPLC) --- p.84 / Chapter 5. --- Discussion and conclusion --- p.91 / Chapter 5.1 --- Genomic studies --- p.91 / Chapter 5.2 --- Presence of adenosine --- p.95 / Chapter 5.3 --- Proteomic --- p.96 / Chapter 5.3.1 --- Protein with increasing expression level --- p.97 / Chapter 5.3.1.1 --- MEI5 (Spot1084) --- p.97 / Chapter 5.3.1.2 --- Hsp70 and hsp60 (Spot 894 & 903) --- p.97 / Chapter 5.3.1.3 --- GRP 78 (Spot 1085) --- p.98 / Chapter 5.3.1.4 --- Ubiquitin (Spot1071) --- p.98 / Chapter 5.3.1.5 --- "Serine-tRNA ligase, glutaminyl-tRNA synthase (Spot 1037, 924)" --- p.99 / Chapter 5.3.1.6 --- 2-isopropylmalate synthase (Spot 862) --- p.99 / Chapter 5.3.1.7 --- Acyl-CoA oxidase 3 (Spot 882) --- p.100 / Chapter 5.3.1.8 --- ATP synthase beta chain (Spot 937) --- p.100 / Chapter 5.3.2 --- Proteins with decreasing expression --- p.101 / Chapter 5.3.2.1 --- 14-3-3 protein (spot 1080) --- p.101 / Chapter 5.3.2.2 --- Actin (Spot 945) --- p.101 / Chapter 5.3.2.3 --- GTP binding protein SPI1 (Spot1031) --- p.102 / Chapter 5.3.2.4 --- Hoclp (Spot 972) --- p.102 / Chapter 5.3.2.5 --- Rchl8p (Spot 983) --- p.103 / Chapter 5.3.2.6 --- Formaldehyde dehydrogenase (Spot 958) --- p.103 / Chapter 5.3.2.7 --- V-type ATPase (Spot 961) --- p.104 / Chapter 5.3.2.8 --- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Spot 987) --- p.104 / Chapter 5.3.2.9 --- Idp3p (Spot 929) --- p.105 / References

Glycogen synthase kinase-3beta (GSK-3beta) has been demonstrated to play a critical role in a diverse range of cellular functions from cell fate determination to cancer development. It is also implicated to be involved in the pathogenesis of neurodegenerative diseases (e.g. Alzheimer's disease), cancers and endocrine disorders (e.g. Type II diabetes). To gain further insight into the cellular mechanisms mediated by GSK-3beta, proteomic approach to identify novel cellular targets has become popular in recent years. GSK-3beta was inhibited by treating with lithium and kenpaullone in SH-SY5Y cell model, and over-expressed in Chinese Hamster Ovary (CHO) Tet-Off cell model. To getting more reliable results, we have used both conventional 2-D approach and Difference Gel Electrophoresis (DIGE) approach for this proteomic study. In 2-D electrophoresis, samples were resolved by two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE). Protein spots were excised from the gels for in-gel trypsin digestion and further subjected to protein identification using mass spectrometry (MALDI-TOF-MS or MS/MS). In the GSK-3beta inhibition approach, cofilin was found to be down-regulated and Pin1 was found to be up-regulated, these consequence events demonstrated inhibition of GSK-3beta would protect the cells from structure alteration and tau hyperphosphorylation. In the GSK-3beta activation approach, cyclin-dependent kinase 5 (CDK-5) was found to significantly up-regulated in tau and GSK-3beta/Tau over-expressed cells, confirmed by Western blotting and RT-PCR. This finding indicates there is a new pathway between GSK-3beta, tau and CDK-5. Pin1 is also identified to be up-regulated after GSK-3beta activation. This result indicated a protection mechanism in response to the accumulation of hyperphosphorylated tau proteins. Our study help to get a better understanding of the GSK-3beta mediated substrates and pathways that help us to identify novel targets for the treatment of neurodegenerative and other diseases mediated by GSK-3beta. / Mak, Ying Cheong. / "September 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4588. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 157-181). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 118-126).

Liquid chromatography-mass spectrometry (LC-MS) based proteomic methods have provided archaeologists with a powerful tool for the discovery and identification of proteins within artifacts. Traditionally, discovery-based methods have utilized a non-targeted full mass scan method in an attempt to identify all proteins present within a given sample. However, increased sensitivity is often needed to target specific proteins in order to test hypotheses. Proteins present within archaeological materials present a unique challenge, as they are often subjected to a variety of chemical transformations both before and after burial. Any preserved proteins will be present within a complex mixture of compounds, and full mass scans often fail to detect less abundant proteins of interest. Consistent and reliable targeted methods are needed to detect protein biomarkers. Taphonomic experimentation was employed as a means to identify the effect of particular processes and conditions on the preservation of mare's milk proteins. In addition, three LC-MS methods were evaluated for their efficiency in identifying mare's milk-specific peptide biomarkers from experimental pottery samples. The ability to reliably detect the presence of these species-specific peptides can help provide evidence about past cultural groups, including the origins of dairying and animal domestication.

Indiana University-Purdue University Indianapolis (IUPUI) / The goal of this study was to determine the effect of a PDK4 (pyruvate dehydrogenase kinase isoenzyme 4) knock-out on mitochondrial protein expression. A 2-D gel based mass spectrometry approach was used to analyze the mitochondrial proteomes of PDK4 wild-type and knockout mice. Mitochondria were isolated from the kidneys of mice in both well-fed and starved states. Previous studies show PDK4 increases greatly in the kidney in response to starvation and diabetes suggesting its significance in glucose homeostasis. The mitochondrial fractions of the four experimental groups (PDK4+/+ fed, PDK4+/+ starved, PDK4-/- fed, and PDK4-/- starved) were separated via large- format, high resolution two-dimensional gel electrophoresis. Gels were scanned, image analyzed, and ANOVA performed followed by a pair-wise multiple comparison procedure (Holm-Sidak method) for statistical analysis. The abundance of a total of 87 unique protein spots was deemed significantly different (p<0.01). 22 spots were up- or down-regulated in the fed knockout vs. fed wild-type; 26 spots in the starved knockout vs. starved wild-type; 61 spots in the fed vs. starved wild-types; and 44 in the fed vs. starved knockouts. Altered protein spots were excised from the gel, trypsinized, and identified via tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins identified with high confidence include ATP synthase proteins, fatty acid metabolism proteins, components of the citric acid cycle and electron transport chain. Proteins of interest were analyzed with Ingenuity Pathway Analysis (IPA) to examine relationships among the proteins and analyze biological pathways, as well as ontological analysis with Generic Gene Ontology (GO) Term Mapper. IPA found a number of canonical pathways, biological functions, and functional networks associated with the 87 proteins. Oxidative phosphorylation was the pathway associated with a majority of the proteins, while the largest network of proteins involved carbohydrate metabolism and energy production. Overall, the effects of starvation were more extensive on mitochondrial protein expression than the PDK4 knockout.