A high-density immunoblotting methodology for quantification of total protein levels and phosphorylation modifications

The components of many signaling pathways have been identified and there is now a need to conduct quantitative data-rich temporal experiments for systems biology and modeling approaches to better understand pathway dynamics and regulation. Here we present a modified Western blotting method that allows the rapid and reproducible quantification and analysis of hundreds of data points per day on proteins and their phosphorylation state at individual sites. The approach is of particular use where samples show a high degree of sample-to-sample variability such as primary cells from multiple donors. We present a case study on the analysis of >800 phosphorylation data points from three phosphorylation sites in three signaling proteins over multiple time points from platelets isolated from ten donors, demonstrating the technique's potential to determine kinetic and regulatory information from limited cell numbers and to investigate signaling variation within a population. We envisage the approach being of use in the analysis of many cellular processes such as signaling pathway dynamics to identify regulatory feedback loops and the investigation of potential drug/inhibitor responses, using primary cells and tissues, to generate information about how a cell's physiological state changes over time.

Serum electrolyte and total protein alterations in Pantaneiro horse during long distance exercise

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Hematological values and total protein of Brasileiro de Hipismo and Breton mares during pregnancy

Estudaram-se as variáveis eritrométricas, leucométricas e a concentração de proteína total (PT) de éguas gestantes das raças Brasileiro de Hipismo (BH) e Bretão. Utilizaram-se 175 éguas sadias, com 3 a 13 anos de idade. Dentre as éguas utilizadas, 89 foram da raça BH, divididas em 14 não-prenhes e 75 prenhes, e 86 éguas da raça Bretão divididas em 11 não-prenhes e 75 prenhes. As éguas foram subdivididas em quatro grupos por raça: grupo controle (éguas não-prenhes, n=14 para BH e n=11 para Bretão); grupo I (primeiro período de gestação, 25-110 dias, n=25 para BH e Bretão), grupo II (segundo período de gestação, 111-210 dias, n=25 para BH e Bretão); e grupo III (terceiro período de gestação, 211-340 dias, n=25 para BH e Bretão). Os valores médios de cada variável eritrométrica, leucométrica e de proteína total foram obtidos para cada raça e, posteriormente, realizou-se comparação entre as mesmas. O número de hemácias (He), volume globular (VG) e teor de hemoglobina (Hb) aumentaram no segundo período de gestação e diminuíram no último período tanto para a raça BH, como para a raça Bretão. Além disso, essas mesmas variáveis apresentaram diferença estatística significativa tanto nas éguas não-prenhas como nas prenhes durante os três períodos gestacionais. Os valores médios obtidos de leucócitos e neutrófilos segmentados aumentaram a partir do segundo período de gestação para as duas raças estudadas.

Glucose, Cholesterol, Total Protein and Growth Factor Insulin-like Type I in Follicular Fluid of River Buffaloes (Bubalus bubalis)

In order to establish the concentrations of glucose, cholesterol, total protein and growth factor insulin-like type I (IGF-I) in the follicular fluid, 26 Murrah breed river buffaloes, between 45 and 70 days postpartum, empty, multiparous, with average live weight of 675 +/- 56 kg and average body condition of 3.5 points on a scale of 1-5, were used in this study. The fluid was collected from dominant follicles with diameters between 8 and 12 mm by OPU, and was not taken into account the stage of the estrous cycle. Using this technique, the wave of follicular development was synchronized six days prior to collection. Biochemical analysis was performed to glucose and cholesterol through the enzymatic colorimetric method using commercial kit glicose CHOLESTEROL GOD-PAP and CHOD-PAP (Kovalent), respectively. Determination of total protein was carried out by using total protein commercial kit (Kovalent) Biuret method, and the readings were performed using absorption spectrophotometry with visible light. Concentration of IGF-I was measured by Radioimmunoassay (RIA) technique using commercial IRMA Kit IGF-I (INMUNOTECH). Descriptive statistics were developed using the PROC MEANS procedure of SAS (2009). Concentration of glucose (4.0 +/- 0.75 mmol / L-1) and IGF-I (340 +/- 129.83 ng / mL (-1)) were higher than those reported by other authors in river buffaloes and cows, respectively. However, cholesterol levels (0.51 +/- 0.12 mmol / L (-1)) and total protein (58.4 +/- 4.43 g / L (-1)) behaved inferior to other studies in same species. The results indicated that there is relationship among the nutritional aspects, diameter of follicles aspirated and productive period in the concentration of biochemical indicators.

Changes in polyamine, total protein and total carbohydrate content and peroxidase activity during the lifetime of chrysanthemum faroe

The aim of the present work was to evaluate the changes in polyamine (PA) content, peroxidase (POX) activity and levels of total protein and total soluble carbohydrates throughout the lifetime of leaves and inflorescences of chrysantemum 'Faroe' treated with gibberellic acidd (GA3) (used in production practices) and kept at room temperature and cold storage. The treatments were composed of four doses of GA3 (0, 15, 30 and 45 mg L-1) applied at the beggining of flower bud formation (28 days after transplanting of seedlings). After harvesting, the stems (95% of the expanded ligule) were stored at 10ºC and 95% relative humidity for 48 hrs, or kept at room temperature. For biochemical analysis samples of leaves and inflorescences were collected at the 4th, 8th, 12th and 16th day after harvest. The application of GA3 in the field and cold storage increased the content of PAs. There was an increase in POX activity in leaves and inflorescences during postharvest and this increase was related to oxidation of the PAs studied. The amount of proteins and carbohydrates in chrysantemum 'Faroe' decreased during the storage at 25ºC and under cold conditions.

MULTIVARIATE STATISTICAL METHOD IN OPTIMIZATION OF PROTEIN EXTRACTION FROM SOYBEAN FLOUR WITH DAIRY WHEY

The aim objective of this project was to evaluate the protein extraction of soybean flour in dairy whey, by the multivariate statistical method with 2(3) experiments. Influence of three variables were considered: temperature, pH and percentage of sodium chloride against the process specific variable ( percentage of protein extraction). It was observed that, during the protein extraction against time and temperature, the treatments at 80 degrees C for 2h presented great values of total protein (5.99%). The increasing for the percentage of protein extraction was major according to the heating time. Therefore, the maximum point from the function that represents the protein extraction was analysed by factorial experiment 2(3). By the results, it was noted that all the variables were important to extraction. After the statistical analyses, was observed that the parameters as pH, temperature, and percentage of sodium chloride, did not sufficient for the extraction process, since did not possible to obtain the inflection point from mathematical function, however, by the other hand, the mathematical model was significant, as well as, predictive.

Global Analysis of Protein Palmitoylation in African Trypanosomes

Many eukaryotic proteins are posttranslationally modified by the esterification of cysteine thiols to long-chain fatty acids. This modification, protein palmitoylation, is catalyzed by a large family of palmitoyl acyltransferases that share an Asp-His-His-Cys Cys-rich domain but differ in their subcellular localizations and substrate specificities. In Trypanosoma brucei, the flagellated protozoan parasite that causes African sleeping sickness, protein palmitoylation has been observed for a few proteins, but the extent and consequences of this modification are largely unknown. We undertook the present study to investigate T. brucei protein palmitoylation at both the enzyme and substrate levels. Treatment of parasites with an inhibitor of total protein palmitoylation caused potent growth inhibition, yet there was no effect on growth by the separate, selective inhibition of each of the 12 individual T. brucei palmitoyl acyltransferases. This suggested either that T. brucei evolved functional redundancy for the palmitoylation of essential palmitoyl proteins or that palmitoylation of some proteins is catalyzed by a noncanonical transferase. To identify the palmitoylated proteins in T. brucei, we performed acyl biotin exchange chemistry on parasite lysates, followed by streptavidin chromatography, two-dimensional liquid chromatography-tandem mass spectrometry protein identification, and QSpec statistical analysis. A total of 124 palmitoylated proteins were identified, with an estimated false discovery rate of 1.0%. This palmitoyl proteome includes all of the known palmitoyl proteins in procyclic-stage T. brucei as well as several proteins whose homologues are palmitoylated in other organisms. Their sequences demonstrate the variety of substrate motifs that support palmitoylation, and their identities illustrate the range of cellular processes affected by palmitoylation in these important pathogens.

Fed State Protein Turnover in Healthy Older Persons under a Usual Protein-Rich Diet

The objective of this study was to verify the protein turnover rates of healthy older persons under a usual protein-rich diet and to compare values to those described in the literature. This cross-sectional study was conducted at Metabolism Unit, Univ. Hospital of the School of Medicine of Ribeirao Preto, Univ. of Sao Paulo, Brazil. In this study, 7 healthy older persons aged 65.4 +/- 2.8 y, with BMI 22.7 +/- 2.4 kg/m(2) and a mean daily protein intake of 1.34 g of protein/kg were studied. A 9-h whole-body (15)N-glycine single-dose study was performed after an overnight fast. During the study, each subject received 6 isoenergetic, isonitrogenous meals at 2-h intervals based on their average intake. Ammonium, urea, and total nitrogen were quantified and analyzed by mass spectrometry, with the determination of total protein turnover rates by the (15)N-glycine method. The results show that total nitrogen output was 3.2 +/- 0.96 g/N and intake 7.7 +/- 1 g/N, (15)N nitrogen flux was 30.6 +/- 6.3 g/9 h. Endogenous nitrogen balance was positive (4.5g +/- g/N in 9 h). In conclusion, the protein turnover of healthy older persons under a usual protein-rich diet is positive during the fed state and has synthesis and degradation rates similar to those previously described in studies involving diet adaptation periods.

Laser phototherapy effect on protein metabolism parameters of rat salivary glands

The aim of this study was to evaluate the effects of infrared diode laser phototherapy (LP) on tissues of the submandibular gland (SMG) and parotid gland (PG). Wistar rats were randomly divided into experimental (A and B) and control (C) groups. A diode laser, 808 nm wavelength, in continuous wave mode, was applied to the PG, SMG and sublingual gland in the experimental groups on two consecutive days. The doses were 4 J/cm(2) and 8 J/cm(2), and total energy was 7 J and 14 J, respectively. The power output (500 mW) and power density (277 mW/cm(2)) were the same for both experimental groups. In order to visualize the area irradiated by the infrared laser, we used a red pilot beam (650 nm) with 3 mW maximum power for the experimental groups. For the control group, the red pilot beam was the only device used. The SMG and PG were removed after 1 week of the first irradiation. Total protein concentration, amylase, peroxidase, catalase and lactate dehydrogenase assays were performed, as well as histological analysis. Statistical tests revealed significant increase in the total protein concentration for groups A and B in the parotid glands (P < 0.05). Based on the results of this study, LP altered the total protein concentration in rats` parotid glands.

Effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) on the viability, proliferation and differentiation of osteoblast-like cells cultured on a chemically modified titanium surface

Aim: The aim of the present study was to assess the influence of the chemical characteristics and roughness of titanium surfaces on the viability, proliferation and differentiation of osteoblast-like cells cultured in a medium supplemented with recombinant human bone morphogenetic protein-7 (rhBMP-7). Material and methods: Osteo-1 cells were grown on titanium disks presenting with the following surfaces: (1) machined, (2) coarse grit-blasted and acid-attacked (SLA) and (3) chemically modified SLA (SLAmod) in the absence or presence of 20 ng/ml rhBMP-7 in culture medium. The viability and number of osteo-1 cells were evaluated after 24 h. Analyses of total protein content (TP) and alkaline phosphatase (AP) activity at 7, 14 and 21 days, collagen content at 7 and 21 days and mineralized matrix formation at 21 days were performed. Results: Cell viability (P=0.5516), cell number (P=0.3485), collagen content (P=0.1165) and mineralized matrix formation (P=0.5319) were not affected by the different surface configurations or by the addition of rhBMP-7 to the medium. Osteo-1 cells cultured on SLA surfaces showed a significant increase in TP at 21 days. The ALPase/TP ratio (P=0.00001) was affected by treatment and time. Conclusion: The results suggest that the addition of rhBMP-7 to the culture medium did not exert any effect on the viability, proliferation or differentiation of osteoblast-like cells grown on the different surfaces tested. All titanium surfaces analyzed allowed the complete expression of the osteoblast phenotype such as matrix mineralization by osteo-1 cells.

Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification

Dissertação apresentada para a obtenção do Grau de Mestre em Biotecnologia, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Development of metabolic labeling strategies to study changes in protein expression

Résumé : Les progrès techniques de la spectrométrie de masse (MS) ont contribué au récent développement de la protéomique. Cette technique peut actuellement détecter, identifier et quantifier des milliers de protéines. Toutefois, elle n'est pas encore assez puissante pour fournir une analyse complète des modifications du protéome corrélées à des phénomènes biologiques. Notre objectif était le développement d'une nouvelle stratégie pour la détection spécifique et la quantification des variations du protéome, basée sur la mesure de la synthèse des protéines plutôt que sur celle de la quantité de protéines totale. Pour cela, nous volions associer le marquage pulsé des protéines par des isotopes stables avec une méthode d'acquisition MS basée sur le balayage des ions précurseurs (precursor ion scan, ou PIS), afin de détecter spécifiquement les protéines ayant intégré les isotopes et d'estimer leur abondance par rapport aux protéines non marquées. Une telle approche peut identifier les protéines avec les plus hauts taux de synthèse dans une période de temps donnée, y compris les protéines dont l'expression augmente spécifiquement suite à un événement précis. Nous avons tout d'abord testé différents acides aminés marqués en combinaison avec des méthodes PIS spécifiques. Ces essais ont permis la détection spécifique des protéines marquées. Cependant, en raison des limitations instrumentales du spectromètre de masse utilisé pour les méthodes PIS, la sensibilité de cette approche s'est révélée être inférieure à une analyse non ciblée réalisée sur un instrument plus récent (Chapitre 2.1). Toutefois, pour l'analyse différentielle de deux milieux de culture conditionnés par des cellules cancéreuses humaines, nous avons utilisé le marquage métabolique pour distinguer les protéines d'origine cellulaire des protéines non marquées du sérum présentes dans les milieux de culture (Chapitre 2.2). Parallèlement, nous avons développé une nouvelle méthode de quantification nommée IBIS, qui utilise des paires d'isotopes stables d'acides aminés capables de produire des ions spécifiques qui peuvent être utilisés pour la quantification relative. La méthode IBIS a été appliquée à l'analyse de deux lignées cellulaires cancéreuses complètement marquées, mais de manière différenciée, par des paires d'acides aminés (Chapitre 2.3). Ensuite, conformément à l'objectif initial de cette thèse, nous avons utilisé une variante pulsée de l'IBIS pour détecter des modifications du protéome dans des cellules HeLa infectée par le virus humain Herpes Simplex-1 (Chapitre 2.4). Ce virus réprime la synthèse des protéines des cellules hôtes afin d'exploiter leur mécanisme de traduction pour la production massive de virions. Comme prévu, de hauts taux de synthèse ont été mesurés pour les protéines virales détectées, attestant de leur haut niveau d'expression. Nous avons de plus identifié un certain nombre de protéines humaines dont le rapport de synthèse et de dégradation (S/D) a été modifié par l'infection virale, ce qui peut donner des indications sur les stratégies utilisées par les virus pour détourner la machinerie cellulaire. En conclusion, nous avons montré dans ce travail que le marquage métabolique peut être employé de façon non conventionnelle pour étudier des dimensions peu explorées en protéomique. Summary : In recent years major technical advancements greatly supported the development of mass spectrometry (MS)-based proteomics. Currently, this technique can efficiently detect, identify and quantify thousands of proteins. However, it is not yet sufficiently powerful to provide a comprehensive analysis of the proteome changes correlated with biological phenomena. The aim of our project was the development of ~a new strategy for the specific detection and quantification of proteomé variations based on measurements of protein synthesis rather than total protein amounts. The rationale for this approach was that changes in protein synthesis more closely reflect dynamic cellular responses than changes in total protein concentrations. Our starting idea was to couple "pulsed" stable-isotope labeling of proteins with a specific MS acquisition method based on precursor ion scan (PIS), to specifically detect proteins that incorporated the label and to simultaneously estimate their abundance, relative to the unlabeled protein isoform. Such approach could highlight proteins with the highest synthesis rate in a given time frame, including proteins specifically up-regulated by a given biological stimulus. As a first step, we tested different isotope-labeled amino acids in combination with dedicated PIS methods and showed that this leads to specific detection of labeled proteins. Sensitivity, however, turned out to be lower than an untargeted analysis run on a more recent instrument, due to MS hardware limitations (Chapter 2.1). We next used metabolic labeling to distinguish the proteins of cellular origin from a high background of unlabeled (serum) proteins, for the differential analysis of two serum-containing culture media conditioned by labeled human cancer cells (Chapter 2.2). As a parallel project we developed a new quantification method (named ISIS), which uses pairs of stable-isotope labeled amino acids able to produce specific reporter ions, which can be used for relative quantification. The ISIS method was applied to the analysis of two fully, yet differentially labeled cancer cell lines, as described in Chapter 2.3. Next, in line with the original purpose of this thesis, we used a "pulsed" variant of ISIS to detect proteome changes in HeLa cells after the infection with human Herpes Simplex Virus-1 (Chapter 2.4). This virus is known to repress the synthesis of host cell proteins to exploit the translation machinery for the massive production of virions. As expected, high synthesis rates were measured for the detected viral proteins, confirming their up-regulation. Moreover, we identified a number of human proteins whose synthesis/degradation ratio (S/D) was affected by the viral infection and which could provide clues on the strategies used by the virus to hijack the cellular machinery. Overall, in this work, we showed that metabolic labeling can be employed in alternative ways to investigate poorly explored dimensions in proteomics.

Regulation of the intracellular distribution, cell surface expression, and protein levels of AMPA receptor GluR2 subunits by the monocarboxylate transporter MCT2 in neuronal cells.

The neuronal monocarboxylate transporter, MCT2, is not only an energy substrate carrier but it is also purported to be a binding partner for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit. To unravel a putative role of MCT2 in the regulation of GluR2 subcellular distribution, Neuro2A cells and primary cultures of mouse cortical neurons were co-transfected with plasmids containing sequences to express the fluorescent proteins mStrawberry (mStb)-fused MCT2 and Venus-fused GluR2. Subsequently, their subcellular distribution was visualized by fluorescence microscopy. GluR2 was led to form perinuclear and dendritic clusters together with MCT2 when co-transfected in Neuro2A cells or in neurons, following the original distribution of MCT2. MCT2 co-transfection had no effect on the intracellular distribution of several other post-synaptic proteins, although it partially affected the intracellular distribution of GluR1 similarly to GluR2. Both cell surface and total protein expression levels of GluR2 were significantly reduced by co-expression with MCT2. Finally, partial perinuclear and dendritic co-localization between MCT2 and Rab8, a member of the small GTPase family involved in membrane trafficking of AMPA receptors, was also observed in co-transfected neurons. These results suggest that MCT2 could influence AMPA receptor trafficking within neurons by modulating GluR2 sorting between different subcellular compartments.