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Characterization of HSP27 Phosphorylation Sites in Human Atherosclerotic Plaque Secretome

Atherosclerosis is one of the main causes of death in developed countries. Atheroma plaque formation is promoted by the interaction between the cells conforming the arterial wall, smooth muscle cells, and endothelial cells, together with lipoproteins and inflammatory cells (mainly ...

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Preparation of Recombinant Adenoviruses

Adenoviruses are viruses with genomes of double-stranded DNA approx 36 kb in size. There are more than 100 different serotypes that have been identified, 47 of them of human origin. Human serotypes have been grouped into six subgenera (A-F) and whereas sequence similarity between genera is low, o ...

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Generation of Recombinant Adeno-Associated Virus

Adeno-associated virus (AAV) was discovered about 30 yr ago as a contaminant of adenovirus preparations. Since its discovery, researchers have described many unique characteristics of AAV biology that have made it attractive as a potential vector for gene therapy. For example, AAV is not pa ...

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Proteomic Identification of S-Nitrosylated Proteins in Endothelial Cells

Nitric oxide (NO) produced in endothelial cells exerts important roles in the vascular system. In recent years, posttranslational modifications induced by NO have been increasingly studied and, among them, cysteine modification by S-nitrosylation (also called S-nitrosation) h ...

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The Proteome and Secretome of Human Arterial Smooth Muscle Cell

Smooth muscle cells (SMCs) play a crucial role in cardiovascular diseases. Proteomic analysis using two-dimensional gel electrophoresis (2DE) associated with mass spectrometry allows characterization of the proteome and secretome of human smooth muscle. The presence of a dist ...

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Real-Time In Vivo Proteomic Identification of Novel Kinase Substrates in Smooth Muscle

Relaxation of smooth muscle can occur through agonists (such as nitric oxide) that activate guanylyl cyclase and stimulate the production of cGMP, activating its target, cGMP-dependent protein kinase (PKG). This kinase can raise the Ca2+ threshold for contraction, thus causing Ca2+ des ...

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Proteomic Analysis of Vascular Smooth Muscle Cells Treated With Ouabain

Apoptosis of vascular smooth muscle cells (VSMC) plays an important role in remodeling the vessel walls, one of the major determinants of long-term blood pressure elevation and an independent risk factor for cardiovascular morbidity and mortality. Apoptosis in VSMC can be inhibited by in ...

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Isolation and Culture of Adult Mouse Cardiac Myocytes

Cardiac myocytes are activated by hormonal and mechanical signals and respond in a variety of ways, from altering contractile function to inducing cardio-protection and growth responses. The use of genetic mouse models allows one to examine the role of cardiac-specific and other genes in c ...

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Proteomic Analysis of Foam Cells

Foam cells are characteristic pathological cells in the lesions of atherosclerosis. Previous works have established macrophage-derived foam cell model to study the central role of the foam cells, and analyzed the protein expression profiles in foam cells. The reported in vitro foam cell ...

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Enrichment of Phosphotyrosine Proteome of Human Platelets by Immunoprecipitation

Proteomics offers the opportunity to comprehensively investigate the anucleate platelet. Here, we present a detailed procedure for enrichment by immunoprecipitation, using the monoclonal antibody 4G10, of the dynamic phosphotyrosine proteome of human platelets. Such an ap ...

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Isolation of the Platelet Releasate

This chapter describes an approach to isolate, separate, and identify the contents of the platelet releasate, a fraction highly enriched for platelet granular and exosomal contents. Investigation into such a fraction will improve our understanding of platelet interactions with ot ...

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Characterization of Circulating Human Monocytes by Proteomic Analysis

We describe a simple method for isolation of human blood monocytes with the high purity required for proteomic analysis, which avoids contamination by other blood cells (platelets and lymphocytes) and the most abundant plasma proteins (albumin and immunoglobulins). Blood monocytes ...

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Cardiovascular Biomarkers Discovery by SELDI-TOF Mass Spectrometry

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (TOF-MS) allows for rapid differential proteomic analysis of various experimental conditions. In the first step of retention chromatography, the proteome to be analyzed is fractionated bas ...

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Detection of Biomarkers of Stroke Using SELDI-TOF

With a mean weight of 1500 g containing around 10 billion neurons, the adult brain represents about 2% of the total body mass, but requires 20% of the total energy produced. It consumes continuously 150 g of glucose and 72 L of oxygen every 24 h. A few minutes interruption of this supply can lead to dramatic brain dam ...

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Depletion of High-Abundance Proteins in Plasma by Immunoaffinity Substraction for Two-Dimensional Difference Gel Electrophoresis Analysis

Blood plasma is believed the most complex human-derived proteome, containing other tissue proteome subsets. Almost all body cells communicate with the plasma, either directly or through tissues or biological fluids, and many of these cells release at least a part of their content into the pl ...

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A Rapid, Economical, and Reproducible Method for Human Serum Delipidation and Albumin and IgG Removal for Proteomic Analysis

Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin (HSA) and immunoglobulin G (IgG) in the serum proteome. Therefore, in order to observe lower-abundance serum pr ...

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Mitoproteome: Human Heart Mitochondrial Protein Sequence Database

The human mitochondrial proteome database has been developed by deriving data from a combination of public repositories and experimental and computational prediction methods. The experimental data is derived from highly purified mitochondria from human heart tissue, whereas ...

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Techniques to Decipher Molecular Diversity by Phage Display

Combinatorial phage display technology may be applied to decipher the molecular diversity of peptide binding specificity to isolated proteins, purified antibodies, cell surfaces, intracellular/cyto-domains, and blood vessels in vivo. The application of such a strategy rang ...

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Congenic/Consomic Models of Hypertension

Human essential hypertension is a complex, multifactorial, quantitative trait under polygenic control. Despite major recent advances in genome sequencing and statistical tools, the genetic dissection of essential hypertension still provides a formidable challenge. Gen ...

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Mouse Knockout Models of Hypertension

Gene-targeting technology allows the planned alteration of any gene in the mouse genome and has been very successfully employed to study the function of numerous gene products in a complete animal. The method includes the design of a suitable targeting construct, its transfection into plu ...

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