Preeclampsia (PE), a life threatening pregnancy-related disorder, is characterized mainly by new onset of hypertension and proteinuria after 20 weeks of gestation. Currently, PE cannot be predicted prior to onset of symptoms and there is no cure for the disease. There is a clear value in having biomarkers able, early in a pregnancy, to identify women at risk for PE so that proper treatment therapies could be developed. Although a number of serum candidate markers have been proposed to be altered in PE patients, their use is limited due to poor sensitivity and specificity. Therefore, there is ongoing need for better set of novel biomarkers predicting PE. Consequently, for my first project, we used a serum proteomic approach involving reversed phase capillary-liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry (cLC-ESI-QTOF). Our approach focuses on the less abundant (nM or lower), lower molecular weight peptides and lipids predicting PE. We got previously collected sera from pregnant women at 12–14 weeks gestation. There were 24 controls, having term uncomplicated pregnancies and 24 cases, which developed PE later in the same pregnancy. Many statistically significant serum PE biomarker candidates were found comparing cases and controls. In addition, multimarker combinations having high detection sensitivity and specificity (AUC >0.9) were developed using logistic regression analysis. For my second project, serum lipidomic analysis of sera from pregnant women was undertaken to determine if useful PE lipid biomarkers exist. A discovery study involving a shotgun lipidomic approach was performed using sera collected at 12-14 weeks of pregnancy from 27 controls with uncomplicated pregnancies and 29 cases that later developed PE. Lipids were extracted using organic solvent and analyzed by direct infusion into a time-of-flight mass spectrometer. Statistically significant lipid markers were found and reevaluated in a second confirmatory study having 43 controls and 37 PE cases. The initial study detected 45 potential PE markers. Of these, 23 markers continued to be statistically significant in the second confirmatory set. Several multi-marker panels with AUC >0.85 and high predictive values were developed from these markers. My third project also involved the above mentioned approach for detection of novel lipid biomarkers for Alzheimer's disease. Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of age-related dementia. Currently, there are no methods to detect Alzheimer's at an early stage when treatment therapies could be applied. Therefore, there is need for detection of panel of biomarkers for detecting patients at risk to AD at an early stage. In the initial discovery set, sera from 29 different stage AD cases and 32 controls were analyzed using direct infusion mass spectrometry (ESI-TOF). This study yielded 89 potential lipid biomarkers which were evaluated in another confirmation study. Of these, 35 markers continued to be statistically significant in the second confirmatory set. Using the confirmed markers, several multi-marker panels with AUC > 0.87 were developed for any stage AD cases vs controls. Multi-marker panels with AUCs > 0.90 were developed for each specific CDR vs controls, including the earliest stage of AD. These lipidomic biomarkers are likely to distinguish AD cases regardless of the stage from controls. In conclusion, we successfully detected, validated and identified low molecular weight novel biomarkers for PE and lipid biomarkers for AD.

Heart failure is a major public health issue, with its prevalence estimated to be 6.5 million adults in the USA. Of the hospitalized heart failure (HF) cases, 50% are characterized by preserved ejection function (HFpEF). In HFpEF, the heart pumps a normal proportion of blood that enters it. However, thickening of the ventricular walls inhibits the chamber filling to normal volume. The direct basis of HFpEF is a slowed elongation of the cardiac muscle during the diastolic phase of the cardiac cycle. Elucidation of mechanisms that mediate relaxation of cardiac muscle could help understand the pathogenic mechanisms in HFpEF. Myocardial contraction and relaxation are tightly controlled processes that involve thick and thin filament regulatory proteins. β-Adrenergic signaling pathway is a major regulator of myocardial contraction and relaxation via the activation of protein kinase A (PKA). Two key myofilament proteins, troponin I (TnI) and myosin binding protein-C (MyBPC), are phosphorylated by PKA following β-adrenergic stimulation. The purpose of this thesis is to develop a liquid chromatography-mass spectrometry (LC-MS) method for the quantification of phosphorylation in TnI and MyBPC and measure the changes in the degree of phosphorylation in transverse-aortic constriction (TAC) mouse hearts, a model representing HFpEF, and sham (control) mouse hearts. The initial approach of the project was to develop a method for quantification of phosphopeptides using synthesized stable isotope labeled (SIL) peptides, both with and without phosphate modification. To accomplish this goal, a multiple reactions monitoring (MRM)-LC-MS method for the quantification of the synthesized SIL peptides was first developed. This method, using low picomole amounts, is applicable to researchers in the field using SIL peptides for quantification. However, when the SIL peptides were actually applied, we determined that there was a selective absorption of some phosphate peptides in the LC column, limiting the use of the SIL peptides for quantification. This result is also of general interest to others trying to identify phosphopeptides, not realizing that some peptides will go unmeasured. Thus, we returned to expanding an earlier method developed in our research group to quantify the degree of phosphorylation. Key to this work was the development of a quantification method directly from heart myofibrillar protein preparations without requiring isolation of individual proteins by gel electrophoresis. Using the LC-MS method developed, we quantified phosphorylation sites of TnI and MyBPC in the TAC and control mouse hearts. The phosphorylation measurements showed no significant difference in phosphorylation between the TAC and control mice, except for one site, S302 in MyBPC that had a 13% decrease in phosphorylation with TAC. We conclude that in our TAC model, PKA dysfunction may not play a role in the initial development of HFpEF.

Protein is an important fraction within bovine milk. This milk protein is not only vital for calf growth and development, but also includes bioactive proteins and peptides that have been shown to enhance the health of animals and humans. Research efforts are focusing on factors, such as nutrition, that can influence the quantity and profile of proteins within the bovine milk proteome. The research outlined herein investigated the impact of diet on the bovine milk proteome. The first experiment examined whether dietary inclusion of grape marc (GM), a condensed tannin (CT) containing by-product from the viticulture industry, could alter the bovine milk proteome through altered nitrogen (N) metabolism. In this experiment, 10 lactating Holstein cows were fed either 2.0 kg dry matter (DM)/ cow/ day of beet pulp: soy hulls in a 50% mixture (control), or 1.5 kg DM/ cow/ day of GM as part of a balanced dairy cow ration for a 28-d trial. Milk samples were obtained for analysis of the high- and low-abundance protein fractions. Skimmed milk samples collected for high-abundance protein analysis were measured using high performance liquid chromatography (HPLC), and liquid-chromatography tandem mass spectrometry (LC-MS/MS) was used to identify proteins in the low-abundance protein enriched fraction. Skimmed milk samples collected for low-abundance milk protein analysis were fractionated and enriched to remove higher abundance proteins. Enriched milk samples were then digested and labeled with isobaric tandem mass tags (TMT) prior to protein identification using LC-MS/MS analysis. There were no changes in the high-abundance protein fraction in response to diet; however, 16 of 127 low-abundance proteins were identified at different relative-abundances due to diet (P ≤ 0.05). While there were no alterations in the metabolic or N status of animals due to GM supplementation, the 12% change in the low-abundance milk protein fraction highlighted the potential for dietary alteration of the bovine milk proteome. A second experiment evaluated the inclusion of alternative forage crops (AFC) as a means to alter the bovine milk proteome. In this experiment, both the skimmed milk and milk fat globule membrane (MFGM) protein fractions were included in analysis. Milk samples were collected from 16 lactating Jersey cattle included in a 21-d grazing experiment, where cows were offered one of two diets. The control group (CON, n=8) grazed a grass-legume pasture mixture containing orchardgrass (Dactylis glomerata), timothy (Phleum pratense), Kentucky bluegrass (Poa pratensis), and white clover (Trifolium repens). The treatment group (AFC, n=8) grazed a similar base pasture that was strip-tilled with oat (Avena sativa), buckwheat (Fagopyrum esculentum), and chickling vetch (Lathyrus sativus) so that the AFC species comprised 10% of the AFC group’s pasture DM intake (DMI). Milk samples were collected for HPLC analysis of the high abundance milk proteins, and LC-MS/MS analysis of the low abundance protein enriched skim milk fraction and MFGM-associated protein fraction. Cows that grazed pastures containing AFC had higher αs1-CAS content (P = 0.005), and higher relative-abundances of 7 low-abundance proteins within the skim milk and MFGM fractions (P ≤ 0.05). While it is plausible that the inclusion of AFC in pasture increased nutrient availability to the mammary gland, the specific mechanisms that could have caused the shifts observed remain unclear. Further investigation is necessary to fully understand the role of diet and the milk protein profile.

The development of traumatic brain injury (TBI) therapeutics and effective translation to clinic remains stubbornly elusive despite the high prevalence of TBI within the United States and across the globe. Interventions must be devised around testable targets, appropriately timed to intercede on secondary results. Here, we have utilized temporal neuroproteomics as an ideal approach to inform on the complex biochemical processing in order to address the well-recognized temporal evolution of TBI pathobiology and interrogate a novel therapeutic target in a mild-moderate rat Controlled Cortical Impact (CCI) within perilesioned somatosensory cortex. First, our findings revealed 2047 proteins significantly impacted within the first two weeks following TBI. Subsequent artificial neural network analysis revealed a delayed-onset cluster of proteins highly enriched in GABAergic neurotransmission and ion transport to reveal the prototypical target potassium/chloride transport 2 (KCC2 or SLC12A5) for further investigation with the KCC2-specific pharmacologic CLP290. Our tested therapeutic window guided by post-translational processing preceding one-day prior to protein loss revealed effective CLP290 restoration of KCC2 localization. We further demonstrated recovery in functional and behavioral assessments with one-day administration paradigm supporting the effectiveness of CLP290 treatment after brain injury. To better understand the underlying mechanism of CLP290, we utilized proteomic and bioinformatic approaches to tease out the biological response to treatment. Results demonstrate recovery of PKCδ-mediated phosphorylation of KCC2 and recovery of transporter activity. Additionally, findings reveal preservation of tyrosine kinase by reversing ubiquitin-mediated proteasomal degradation. Our functional assessment of secondary injury insults two-weeks following TBI revealed recovery in seizure threshold, reduction in lesion expansion and a decrease in cell loss suggesting maintained recovery of KCC2 and restored E/I balance. In conclusion, the presented studies in these two chapters propose a novel approach for development of therapeutics for TBI and test the selective manipulation via pharmacological intervention. These findings are promising for the development and treatment of other neurological disorders.

O carcinoma de células renais (CCR) representa 3% das neoplasias humanas e aproximadamente 90% das neoplasias renais e entre os tumores urológicos. O CCR é bastante resistente à radioterapia convencional. Entretanto, com o aparecimento de novas técnicas/equipamentos é possivel a aplicação de doses com precisão presevando-se os tecidos adjacentes. Para a verificação da expressão proteica em diferentes tecidos e fluidos corporais, foi utilizado o estudo proteômico sob diferentes condições e / ou tempos. A espectrometria de massa permite a identificação e quantificação de milhares de proteínas e peptídeos em fluidos biológicos ou células lisadas, sendo assim uma ferramenta poderosa para identificação de potenciais biomarcadores de doenças. A finalidade deste trabalho foi analisar o perfil proteico das células de adenocarcinoma renal (786-0) após a radiação com doses que variaram de 2 a 10 Gy. Os dados foram tratados com o programa One-way ANOVA seguida do de Bonferroni. Pelo ensaio de clonogenicidade definiou-se a dose de 8 Gy como a ideal estudos. A extração das proteínas citoplasmáticas foi realizada com o kit de extração do proteoma subcelular PE e a quantificação das proteínas feita pelo método de Lowry. A integridade das proteínas foi analisada por SDS-PAGE e a solução proteica foi verificada em LTQ Orbitrap. Os resultados gerados foram analisados pelo servidor MASCOT para a busca de peptídeos. A análise por espectrometria de massa foi possível identificar 44 proteínas nas amostras não irradiadas - e 87 das amostras irradiadas. Nas amostras não irradiadas a distribuição dos grupos funcionais foi de síntese proteica 46,66%; Metabolismo energético 16,66%; Migração e proliferação 16,66%; Antioxidantes 3,33%. No grupo irradiado síntese proteica 35,89%; Metabolismo energético 20,51%; Migração e proliferação 20,51%; Antioxidantes 5,12%; Chaperonas moleculares 5,12% e Endopeptidases 5,12%. Em seguida, analisou-se o espetro de as sequências com escores acima de 40. Nas amostras irradiadas encontrou-se: ENO1 (47 kDa); A VIM (53 kDa)/ HEL113; PSMA1; TRAJ56; hCG; Cofilina-1 (19 kDa); HIST1H4H; PKM2; ANXA1; HSPB1/ HSP27. Deste grupo, entendemos que a subunidade alfa do proteassoma - tipo 1 (PSMA1), que possui uma atividade molecular de endopeptidase, seja um alvo interessante para estudos posteriores. / Renal cell carcinoma (CRC) represents 3% of human neoplasms and approximately 90% of renal neoplasms and among urological tumors. The RCC is quite resistant to conventional radiotherapy. This technique allows the dose of radiation, in a single fraction, to be accurately applied to the tumor and the tissues adjacent to it, most of the time, are spared. The protein expression analysis in different tissues and body fluids was realized according the proteomics study under different conditions and / or times. Mass spectrometry allows the identification and quantification of thousands of proteins and peptides in a biological fluid or lysed cells, and is analyzed on a platform to identify differences in the expression of proteins associated with the proliferation of cancer cells and to establish potential biomarkers predictive of answer. The aim of this work was to analyze the protein profile of human renal cancer 786-0 cells after radiation, evaluated according the mitotic potential of irradiated cells in GammaCell performed at doses of 2 to 10 Gy. The irradiated cell colonies were stained and counted, and the multiple comparisons were analyzed by One-way ANOVA followed by the Bonferroni post-test. The dose of 8 Gy was defined as ideal for cell irradiation, the cytoplasmic proteins were extracted by the subcellular proteome PE kit and quantified by the Lowry method. For protein integrity analysis, SDS-PAGE was performed. The protein solution was analyzed in LTQ Orbitrap. The generated result was analyzed by the MASCOT server for search of peptides. Mass spectrometric analysis identified 44 proteins in non-irradiated samples - and 87 in irradiated samples. In non-irradiated samples the distribution of functional groups was protein synthesis 46.66%; Energy metabolism 16.66%; Migration and proliferation 16.66%; Antioxidants 3.33%. In the irradiated group protein synthesis 35.89%; Energy metabolism 20.51%; Migration and proliferation 20.51%; Antioxidants 5.12%; Molecular chaperones 5.12% and Endopeptidases 5.12%. Then, the spectrum of the sequences above 40 was analyzed. In the irradiated samples we found: ENO1 (47 kDa); VIM (53 kDa) / HEL113; PSMA1; TRAJ56; hCG; Cophylline-1 (19 kDa); HIST1H4H; PKM2; ANXA1; HSPB1 / HSP27. From this group, we identified the alpha subunit of the proteome type 1 (PSMA1) that has an endopeptidase This group, understood as a proteasome - type 1 (PSMA1) alpha subunit, has an endopeptidase molecular molecule and is an interesting target for further studies.

Vibrio parahaemolyticus is a leading cause of seafood-borne illness with a newly emerged pandemic strain. Previous studies compared the pandemic and non-pandemic strains to understand the evolution of the pandemic strain but no definitive explanation for its emergence has been discovered. This study investigated the molecular characteristics of the pandemic strain and growth characteristics at different temperatures. The hypothesis tested was that pandemic strains of V. parahaemolyticus have modifications to their proteome that give a selective advantage over the other V. parahaemolyticus strains at temperatures normally encountered in the environment. Molecular typing techniques; automated ribotyping, pandemic specific PCR and multilocus sequence typing (MLST), were compared to determine the best method for pandemic strain determination. MLST was the best method because it was the most informative and accurate. Furthermore, nine Florida outbreak strains were identified as pandemic. Using representatives of both strains, growth curves were produced at four temperatures. The five pandemic strains had a significantly faster growth rate at 12°C than five non-pandemic strains. Temperature specific proteomic comparisons were completed using liquid chromatography followed by tandem mass spectroscopy. The proteome differences between these two groups at 12°C included three proteins (DnaA, DnaJ-related protein and DnaK-related protein) with functions related to cold stress. DnaA was expressed in the non-pandemic strain and not the pandemic strain, while the reverse was true for DnaJ-related and DnaK-related proteins. Western blot analysis and LC-MS/MS analysis on additional strains did not support the initial LC-MS/MS results. Growth studies using expression recombinants were employed to investigate these proteins on growth at 12°C. The overexpression of DnaA and DnaJ-related proteins did not significantly alter the growth rates compared to the control strain, but the overexpression recombinant strains DnaK-5 has a significantly slower growth rate than the control strain, the opposite direction as expected. The pandemic strain grows faster at lower temperatures, but the reason has not been determined. A theory is offered in which the pandemic growth advantage related to regulation of cold stress, leading to a shorter lag phase and faster growth rate after acclimation to the lower temperatures. Further experiments to investigate this theory are discussed.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

<p>The sequencing of genomes has caused a growing demand for functional analysis of gene products. This research field named proteomics is derived from the term proteome, which by analogy to genome is defined as all proteins expressed by a cell or a tissue. Proteomics is however methodologically restricted to the analysis of proteins with higher molecular weights. The development of a technology which includes peptides with low molecular weight and small proteins is needed, since peptides play a central role in many biological processes. </p><p>To study endogenous peptides and hormones, the peptidome, an improved method comprising rapid deactivation in combination with nano-flow liquid chromatography (LC) and mass spectrometry (MS) was developed. The method has been used to investigate endogenous peptides in brains of mouse and rat. Several novel peptides have been discovered together with known neuropeptides. </p><p>To elucidate the <i>post mortem</i> time influence on peptides and proteins, a time course study was performed using peptidomics and proteomics technologies. Already after three minutes a substantial amount of protein fragments emerged in the peptidomics study and some endogenous peptides were drastically reduced with increasing <i>post mortem</i> time. Of about 1500 proteins investigated, 53 were found to be significantly changed at 10 minutes <i>post mortem</i> as compared to control. Moreover, using western blot the level of MAPK phosphorylation was shown to decrease by 95% in the 10 minutes <i>post mortem </i>sample. </p><p>A database, SwePep (a repository of endogenous peptides, hormones and small proteins), was constructed to facilitate identification using MS. The database also contains additional information concerning the peptides such as physical properties. A method for analysis of LC-MS data, including scanning for, and further profiling of, biologically significant peptides was developed. We show that peptides present in different amounts in groups of samples can be automatically detected.</p><p>The peptidome approach was used to investigate levels of peptides in two animal models of Parkinson’s disease. PEP-19, was found to be significantly decreased in the striatum of MPTP lesioned parkinsonian mice. The localization and expression was further investigated by imaging MALDI MS and by <i>in situ</i> hybridization. The brain peptidome of reserpine treated mice was investigated and displayed a number of significantly altered peptides. This thesis demonstrates that the peptidomics approach allows for the study of complex biochemical processes.</p>

Fueled by ever-growing genomic information and rapid developments of proteomics–the large scale analysis of proteins and mapping its functional role has become one of the most important disciplines for characterizing complex cell function. For building functional linkages between the biomolecules, and for providing insight into the mechanisms of biological processes, last decade witnessed the exploration of combinatorial and chip technology for the detection of bimolecules in a high throughput and spatially addressable fashion. Among the various techniques developed, the protein chip technology has been rapid. Recently we demonstrated a new platform called “Spacially addressable protein array” (SAPA) to profile the ligand receptor interactions. To optimize the platform, the present study investigated various parameters such as the surface chemistry and role of additives for achieving high density and high-throughput detection with minimal nonspecific protein adsorption. In summary the present poster will address some of the critical challenges in protein micro array technology and the process of fine tuning to achieve the optimum system for solving real biological problems. / Singapore-MIT Alliance (SMA)

The field of proteomics encompasses the study of identities, interactions, and dynamics of all proteins expressed by a living system. Research in this dissertation blends biochemical and quantitative proteomics techniques to increase the latitude of biological applications for the bottom-up mass spectrometry proteomics approach. Together, isolation of selected protein “targets,” such as multiprotein complexes, and quantitative characterization yields information essential for more detailed understanding of microbial cell function. Often, a challenging aspect of characterizing a variety of biochemically enriched samples is limited protein yield. This dissertation describes an enzymatic proteolysis protocol employing an organic/aqueous solvent that alleviates excessive handling steps to reduce losses during sample preparation for small quantities of protein samples. Presence of artifactual, non-specific proteins in enriched protein complex isolates complicates biological interpretation of specific protein interactions. Heterologous expression of affinity-tagged bait proteins may also cause unintended collateral effects. A series of local and global protein isotope ratio measurements were performed to differentiate authentic interactions from artifactual interactions among affinity-isolated complexes and assess collateral effects, respectively. Protein localization provides clues regarding protein function. To infer protein localization, quantitative proteomics techniques were used to estimate protein enrichment of cold osmotic shock periplasmic isolates. Protein isotope ratios indicating enrichment, combined with identification of amino-terminal signal peptide cleavages, increase confidence of periplasmic localization. Collectively, this dissertation provides a framework for tailoring biochemical and quantitative techniques for targeted characterization of microbial protein isolates.