Transforming growth factor-β and inflammation in vascular (type IV) Ehlers-Danlos syndrome.

Background: Vascular Ehlers-Danlos syndrome (VEDS) causes reduced life expectancy because of arterial dissections/rupture and hollow organ rupture. Although the causative gene, COL3A1, was identified >20 years ago, there has been limited progress in understanding the disease mechanisms or identifying treatments.

Methods And Results: We studied inflammatory and transforming growth factor-β (TGF-β) signaling biomarkers in plasma and from dermal fibroblasts from patients with VEDS.

Cardiac resynchronization sensitizes the sarcomere to calcium by reactivating GSK-3β.

Cardiac resynchronization therapy (CRT), the application of biventricular stimulation to correct discoordinate contraction, is the only heart failure treatment that enhances acute and chronic systolic function, increases cardiac work, and reduces mortality. Resting myocyte function also increases after CRT despite only modest improvement in calcium transients, suggesting that CRT may enhance myofilament calcium responsiveness. To test this hypothesis, we examined adult dogs subjected to tachypacing-induced heart failure for 6 weeks, concurrent with ventricular dyssynchrony (HF(dys)) or CRT.

Immune-mediated pore-forming pathways induce cellular hypercitrullination and generate citrullinated autoantigens in rheumatoid arthritis.

Autoantibodies to citrullinated protein antigens are specific markers of rheumatoid arthritis (RA). Although protein citrullination can be activated by numerous stimuli in cells, it remains unclear which of these produce the prominent citrullinated autoantigens targeted in RA. In these studies, we show that RA synovial fluid cells have an unusual pattern of citrullination with marked citrullination of proteins across the broad range of molecular weights, which we term cellular hypercitrullination.

PKCα-specific phosphorylation of the troponin complex in human myocardium: a functional and proteomics analysis.

Aims: Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.

Identification and characterization of citrulline-modified brain proteins by combining HCD and CID fragmentation.

Citrullination is a protein PTM of arginine residues catalyzed by peptidylarginine deiminase. Protein citrullination has been detected in the CNS and associated with a number of neurological diseases. However, identifying citrullinated proteins from complex mixtures and pinpointing citrullinated residues have been limited.

Can proteomics yield insight into aging aorta?

The aging aorta exhibits structural and physiological changes that are reflected in the proteome of its component cells types. The advance in proteomic technologies has made it possible to analyze the quantity of proteins associated with the natural history of aortic aging. These alterations reflect the molecular and cellular mechanisms of aging and could provide an opportunity to predict vascular health.

The Human Eye Proteome Project: perspectives on an emerging proteome.

There are an estimated 285 million people with visual impairment worldwide, of whom 39 million are blind. The pathogenesis of many eye diseases remains poorly understood. The human eye is currently an emerging proteome that may provide key insight into the biological pathways of disease.

Characterization of the cardiac myosin binding protein-C phosphoproteome in healthy and failing human hearts.

Introduction: Cardiac myosin binding protein-C (cMyBP-C) becomes dephosphorylated in the failing heart and reduced phosphorylation-dependent regulation of cMyBP-C has been implicated in contractile dysfunction. To date, several phosphorylation sites have been identified for human cMyBP-C; however, a comprehensive characterization of the cMyBP-C phosphoproteome is lacking. This study aimed to characterize the cMyBP-C phosphoproteome using two different proteomic-based methods in explanted donor and end-stage failing hearts.

Optimized method for identification of the proteomes secreted by cardiac cells.

In the past, various studies using different methods have been carried out to analyze proteins secreted by cells. There are several crucial steps that have to be followed to ensure successful secreted proteome detection and identification. Simultaneously with the optimization of the experimental conditions for various cell type culturing and subsequent cell conditioning to obtain conditioned medium with secreted proteins in vitro, the analytical separation methods for fractionation of complex protein mixture and mass spectrometry for protein identification are of high importance.

Using pure protein to build a multiple reaction monitoring mass spectrometry assay for targeted detection and quantitation.

Multiple reaction monitoring (MRM) is an increasingly popular mass spectrometry-based method to simultaneously detect and quantify multiple proteins. MRM is particularly useful for validating biomarkers discovered with a mass spectrometer and any analite discovered by MS can be monitored by MR because an MRM assay can be developed without the need to generate specific antibodies. In this chapter, we present a robust and systematic procedure to rapidly build a high-sensitivity MRM assay using purified protein as the starting material.

Quantification of mitochondrial S-nitrosylation by CysTMT⁶ switch assay.

S-nitrosylation (SNO) is an important oxidative posttranslational modification in the regulation of cardiac mitochondria. SNO modification of several mitochondrial proteins has been associated with cardiac preconditioning and improved cell survival following ischemia/reperfusion injury. Due to their labile nature, SNO modifications are challenging to study using traditional biochemical techniques; particularly, the identification of individual modified cysteine residues.

Constrained selected reaction monitoring: quantification of selected post-translational modifications and protein isoforms.

Selected reaction monitoring (SRM) is a mass spectrometry method that can target signature peptides to provide for the detection and quantitation of specific proteins in complex biological samples. When quantifying a protein, multiple peptides are generated using a specific protease such as trypsin, thereby allowing a choice of signature peptides with robust signals. In contrast, signature peptide selection can be constrained when the goal is to monitor a specific post-translational modification (PTM) or protein isoform, as the signature peptide must include the amino acid residue(s) of PTM attachment or sequence variation.

Improved protein extraction and protein identification from archival formalin-fixed paraffin-embedded human aortas.

Purpose: Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formalin-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics.

Experiment Design: Proteins were extracted from fresh frozen aorta at room temperature (RT). FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure.

Cysteine oxidative posttranslational modifications: emerging regulation in the cardiovascular system.

In the cardiovascular system, changes in oxidative balance can affect many aspects of cellular physiology through redox-signaling. Depending on the magnitude, fluctuations in the cell's production of reactive oxygen and nitrogen species can regulate normal metabolic processes, activate protective mechanisms, or be cytotoxic. Reactive oxygen and nitrogen species can have many effects including the posttranslational modification of proteins at critical cysteine thiols.

Redox regulation of mitochondrial ATP synthase.

Reversible cysteine oxidative post-translational modifications (Ox-PTMs) represent an important mechanism to regulate protein structure and function. In mitochondria, redox reactions can modulate components of the electron transport chain (ETC), the F(1)F(0)-ATP synthase complex, and other matrix proteins/enzymes. Emerging evidence has linked Ox-PTMs to mitochondrial dysfunction and heart failure, highlighting some potential therapeutic avenues.

A fast and reproducible method for albumin isolation and depletion from serum and cerebrospinal fluid.

Analysis of serum and plasma proteomes is a common approach for biomarker discovery, and the removal of high-abundant proteins, such as albumin and immunoglobins, is usually the first step in the analysis. However, albumin binds peptides and proteins, which raises concerns as to how the removal of albumin could impact the outcome of the biomarker study while ignoring the possibility that this could be a biomarker subproteome itself. The first goal of this study was to test a new commercially available affinity capture reagent from Protea Biosciences and to compare the efficiency and reproducibility to four other commercially available albumin depletion methods.

The biology/disease-driven human proteome project (B/D-HPP): enabling protein research for the life sciences community.

The biology and disease oriented branch of the Human Proteome Project (B/D-HPP) was established by the Human Proteome Organization (HUPO) with the main goal of supporting the broad application of state-of the-art measurements of proteins and proteomes by life scientists studying the molecular mechanisms of biological processes and human disease. This will be accomplished through the generation of research and informational resources that will support the routine and definitive measurement of the process or disease relevant proteins. The B/D-HPP is highly complementary to the C-HPP and will provide datasets and biological characterization useful to the C-HPP teams.

Chasing cysteine oxidative modifications: proteomic tools for characterizing cysteine redox status.

Redox-proteomics involves the large scale analysis of oxidative protein post-translational modifications. In particular, cysteine residues have become the subject of intensifying research interest because of their redox-reactive thiol side chain. Certain reactive cysteine residues can function as redox-switches, which sense changes in the local redox-environment by flipping between the reduced and oxidized state.