Quantitative analysis of myofilament protein phosphorylation in small cardiac biopsies

Phosphorylation of cardiac myofilament proteins represents one of the main post‐translational mechanisms that regulate cardiac pump function. Human studies are often limited by the amount of available tissue as biopsies taken during cardiac catheterization weigh only 1 mg (dry weight). Similarly, investigation of time‐ (or dose‐) dependent changes in protein phosphorylation in animal studies is often hampered by tissue availability. The present study describes quantitative analysis of phosphorylation status of multiple myofilament proteins by 2‐DE and Pro‐Q® Diamond stained gradient gels using minor amounts (?0.5 mg dry weight) of human and pig cardiac tissue.     

Top-down mass spectrometry of cardiac myofilament proteins in health and disease

Myofilaments are composed of thin and thick filaments that coordinate with each other to regulate muscle contraction and relaxation. PTMs together with genetic variations and alternative splicing of the myofilament proteins play essential roles in regulating cardiac contractility in health and disease. Therefore, a comprehensive characterization of the myofilament proteins in physiological and pathological conditions is essential for better understanding the molecular basis of cardiac function and dysfunction. Due to the vast complexity and dynamic nature of proteins, it is challenging to obtain a holistic view of myofilament protein modifications. In recent years, top-down MS has emerged as a powerful approach to study isoform composition and PTMs of proteins owing to its advantage of complete sequence coverage and its ability to identify PTMs and sequence variants without a priori knowledge. In this review, we will discuss the application of top-down MS to the study of cardiac myofilaments and highlight the insights it provides into the understanding of molecular mechanisms in contractile dysfunction of heart failure. Particularly, recent results of cardiac troponin and tropomyosin modifications will be elaborated. The limitations and perspectives on the use of top-down MS for myofilament protein characterization will also be briefly discussed.     

Cardiac myofilaments: from proteome to pathophysiology

This review addresses the functional consequences of altered post-translational modifications of cardiac myofilament proteins in cardiac diseases such as heart failure and ischemia. The modifications of thick and thin filament proteins as well as titin are addressed. Understanding the functional consequences of altered protein modifications is an essential step in the development of targeted therapies for common cardiac diseases.     

Proteomics in paediatric cardiac surgery: Is a personalised approach feasible?

The incidence of congenital cardiac abnormalities remains high. Paediatric patients with congenital cardiac defects often require surgery at a young age. The surgeries are often long and complex, rendering this population particularly vulnerable to the deleterious effects of cardiopulmonary bypass and cardiac surgery. The search for cardioprotective strategies is ongoing in an attempt to reduce the morbidity in this population. In the post-genomic era, it is apparent that simply determining the genomic sequences holds little diagnostic potential and means to determine progression of disease and response to treatment. The field of proteomics is expanding and application of proteomic techniques in the clinical setting holds great potential to advance our understanding of the proteomic changes involved in specific disease stages. This review will assess the application of proteomic techniques in the setting of paediatric cardiac surgery and highlight the need to obtain a clear understanding of the role of various proteins in children with cardiac conditions. The success and challenges of the available proteomic technology will be discussed as well as the future potential of proteomic methods for advancing our understanding of protein changes in children requiring cardiac surgery.     

Targeted proteomics of myofilament phosphorylation and other protein posttranslational modifications

Global cardiac myofilament protein phosphorylation levels, and their site-specific stoichiometry, are physiologically and clinically relevant for heart function. Unlike myofilament phosphorylation, other PTMs such as O-GlcNAcylation are just beginning to gain attention due to their potential physiological and clinical implications. This review will focus on what is currently known about cardiac troponin I phosphorylation, and on the potential physiological and clinical impact of targeted proteomics including new findings on cardiac troponin I sites and stoichiometry. We will then discuss the increasing recognition of other myofilament PTMs functional relevance and the potential of targeted MS approaches, particularly MRM, for accelerating their systematic characterization. In addition, we will broadly discuss the development and application of MRM to quantitatively assess site-specific PTMs. Finally, we will give an overview of expert's consensus on MRM methods design/validation and best practices to develop MRM assays intended to reach clinical application. The unique ability of MRM and similar methods to identify and quantify cardiac myofilament PTMs is likely to become central in answering important biological questions in the field of cardiac integrative physiology.     

Characterization of proteomic changes in cardiac mitochondria in streptozotocin-diabetic rats using iTRAQ™ isobaric tags

Diabetes now affects more than 5% of the world's population and heart failure is the most common cause of death amongst diabetic patients. Accumulating evidence supports a view that myocardial mitochondrial structural and functional changes are central to the onset of diabetic heart failure, but the exact nature of these changes at the proteomic level remains unclear.Here we report on proteomic changes in diabetic rat heart mitochondria following 120 days of streptozotocin‐diabetes using the recently developed iTRAQ? labeling method, which permits quantification of proteins directly from complex mixtures, bypassing the limitations associated with gel‐based methods such as 2‐DE. Of 252 unique proteins identified, 144 were represented in at least three of six individual paired experiments. Relative amounts of 65 proteins differed significantly between the groups, confirming that the cardiac mitochondrial proteome is indeed impacted by diabetes. The most significant changes were increased protein levels of enzymes involved in mitochondrial oxidation of long‐chain fatty acids, which was also confirmed by enzyme assays, and decreased levels of multiple enzymes involved in oxidative phosphorylation and catabolism of short‐chain fatty acids and branched‐chain amino acids. We also found significant changes in levels of several enzymes linked to oxidative stress.     

Shotgun MS proteomic analysis of bronchoalveolar lavage fluid in normal subjects

We provide a review of proteomic techniques used to characterize the bronchoalveolar lavage fluid (BALF) proteome of normal healthy subjects. Bronchoalveolar lavage (BAL) is the most common technique for sampling the components of the alveolar space. The proteomic techniques used to study normal BALF include protein separation by 2DE, whereby proteins were identified by comparison to a reference gel as well as high pressure liquid chromatography (HPLC)-MS/MS, also known as shotgun proteomics. We summarize recent progress using shotgun MS technologies to define the normal BALF proteome. Surprisingly, we find that despite advances in shotgun proteomic technologies over the course of the last 10 years, which have resulted in greater numbers of proteins being identified, the functional landscape of normal BALF proteome was similarly described by all methods examined.     

Annexin A4: A novel molecular marker for gastric cancer with Helicobacter pylori infection using proteomics approach

Helicobacter pylori was reported to be an important risk factor for the carcinogenesis of gastric cancer. Here, we used a proteomic approach to find differentially expressed proteins between the normal and tumor tissue of gastric cancer patients infected with H. pylori. In our results, we found annexin A4 was over-expressed in patients infected with H. pylori and was found in tumor cells, and over-expressed in gastric cancer SCM-1 cells after H. pylori infection. Ca(2+ ) can be induced by H. pylori and interact with annexin A4 Ca(2+) binding site to block the calmodulin-activated chloride conductance activation; therefore, it produces a new environment that benefits the malignant existence of H. pylori and raises the risk for gastric cancer. We also found interleuken-8 (IL-8) expression levels were increased in H. pylori infected SCM-1 cells. Combined with previous reports and our results, we summarize that the over-expression of annexin A4 in SCM-1 cells with H. pylori infection may subsequently induce IL-8 which can further cause tumor angiogenesis. In this paper, we show that annexin A4 is a potential novel molecular marker for gastric cancer with H. pylori infection, and our results may provide a new insight in the development of new anti-cancer drugs.     

Identification and validation of calmodulin as a binding protein of an anti-proliferative small molecule 3,4-dihydroisoquinolinium salt

IHY-153 (2-(2,5-difluorobenzyl)-3,4-dihydro-5-(10-hydroxydecyl)-6-methoxy-1-undecylisoquinolinium bromide) was recently discovered as a small molecule that potently inhibits proliferation of tumor cells by inducing cell-cycle arrest at G0-G1 phase. To investigate the basis of anti-proliferative activity of IHY-153, cellular binding proteins of biotinyl-IHY-153 were screened using T7 phage displayed human cDNA libraries. Calmodulin-expressing phage specifically bound to immobilized IHY-153 in a Ca(2+) -dependent manner. The interaction between IHY-153 and Ca(2+) /CaM was validated through phage competition binding assays, surface plasmon resonance analysis, and molecular modeling. IHY-153 induced sustained phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and subsequently increased p21(WAF1) expression in colon cancer cells. These results demonstrate that IHY-153, a novel small molecule, targets Ca(2+) /CaM and indicate that this compound functions as an anti-proliferative agent by influencing Ca(2+) /CaM-dependent signal transduction.     

Neuromodulation of short-term synaptic dynamics examined in a mechanistic model based on kinetics of calcium currents

Network plasticity arises in large part due to the effects of exogenous neuromodulators. We investigate the neuromodulatory effects on short-term synaptic dynamics. The synapse from the lateral pyloric (LP) to the pyloric dilator (PD) neuron in the pyloric network of the crab C. borealis has both spike-mediated and non-spike-mediated (graded) components. Previous studies have shown that the graded component of this synapse exhibits short-term depression. Recent results from our lab indicate that in the presence of neuromodulatory peptide proctolin, low-amplitude presynaptic stimuli switch the short-term dynamics of this graded component from depression to facilitation. In this study, we show that this facilitation is correlated with the activation of a presynaptic inward current that is blocked by Mn(2+) suggesting that it is a slowly-accumulating Ca(2+) current. We modify a mechanistic model of synaptic release by assuming that the low-voltage-activating Ca(2+) current in our system is composed of two currents with fast (I(CaF)) and slow (I(CaS)) kinetics. We show that if proctolin adjusts the activation rate of I(CaS), this leads to accumulation of local intracellular Ca(2+) in response to multiple presynaptic voltage stimuli which, in turn, results in synaptic facilitation. Additionally, we assume that proctolin increases the maximal conductances of Ca(2+) currents in the model, consistent with the increased synaptic release found in the experiments. We find that these two presynaptic actions of proctolin in the model are sufficient to describe its actions on the short-term dynamics of the LP to PD synapse.     

Colorectal cancer proteomics, molecular characterization and biomarker discovery

Colorectal cancer (CRC) is a widespread disease, whose major genetic changes and mutations have been well characterized in the sporadic form. Much less is known at the protein and proteome level. Still, CRC has been the subject of multiple proteomic studies due to the urgent necessity of finding clinically relevant markers and to elucidate the molecular mechanisms underlying the progression of the disease. These proteomic approaches have been limited by different technical issues, mainly related with sensitivity and reproducibility. However, recent advances in proteomic techniques and MS systems have rekindled the quest for new biomarkers in CRC and an improved molecular characterization. In this review, we will discuss the application of different proteomic approaches to the identification of differentially expressed proteins in CRC. In particular, we will make a critical assessment about the use of 2-D DIGE, MS and protein microarray technologies, in their different formats, to identify up- or downregulated proteins and/or autoantibodies profiles that could be useful for CRC characterization and diagnosis. Despite a wide list of potential biomarkers, it is clear that more scientific efforts and technical advances are still needed to cover the range of low-abundant proteins, which may play a key role in CRC diagnostics and progression.     

Pilot proteomic analysis of cerebrospinal fluid in Alzheimer's disease

Proteomic analysis of cerebrospinal fluid (CSF) holds great promise in understanding the progression of neurodegenerative diseases, including Alzheimer's disease (AD). As one of the primary reservoirs of neuronal biomolecules, CSF provides a window into the biochemical and cellular aspects of the neurological environment. CSF can be drawn from living participants allowing the potential alignment of clinical changes with these biochemical markers. Using cutting-edge mass spectrometry technologies, we perform a streamlined proteomic analysis of CSF. We quantify greater than 700 proteins across 10 pairs of age- and sex-matched participants in approximately one hour of analysis time each. Using the paired participant study structure, we identify a small group of biologically relevant proteins that show substantial changes in abundance between cognitive normal and AD participants, which were then analyzed at the peptide level using parallel reaction monitoring experiments. Our findings suggest the utility of fractionating a single sample and using matching to increase proteomic depth in cerebrospinal fluid, as well as the potential power of an expanded study.     

Redox signalling in cardiovascular disease

Oxidative stress has almost universally and unequivocally been implicated in the pathogenesis of all major diseases, including those of the cardiovascular system. Oxidative stress in cells and cardiovascular biology was once considered only in terms of injury, disease and dysfunction. However, it is now appreciated that oxidants are also produced in healthy tissues, and they function as signalling molecules transmitting information throughout the cell. Conversely, when cells move to a more reduced state, as can occur when oxygen is limiting, this can also result in alterations in the function of biomolecules and subsequently cells. At the centre of this 'redox signalling' are oxidoreductive chemical reactions involving oxidants or reductants post translationally modifying proteins. These structural alterations allow changes in cellular redox state to be coupled to alterations in cell function. In this review, we consider aspects of redox signalling in the cardiovascular system, focusing on the molecular basis of redox sensing by proteins and the array of post-translational oxidative modifications that can occur. In addition, we discuss studies utilising proteomic methods to identify redox-sensitive cardiac proteins, as well as those using this technology more broadly to assess redox signalling in cardiovascular disease.     

Differential regulation of redox proteins and chaperones in HbEβ-thalassemia erythrocyte proteome

Purpose: In (hemoglobin, Hb) HbEβ‐thalassemia, HbE (β‐26 Glu→Lys) interacts with β‐thalassemia to produce clinical manifestation of varying severity. This is the first proteomic effort to study changes in protein levels of erythrocytes isolated from HbEβ‐thalassemic patients compared to normal. Experimental design: We have used 2‐DE and MALDI‐MS/MS‐based techniques to investigate the differential proteome profiling of membrane and Hb‐depleted fraction of cytosolic proteins of erythrocytes isolated from the peripheral blood samples of HbEβ‐thalassemia patients and normal volunteers. Results: Our study showed that redox regulators such as peroxiredoxin 2, Cu‐Zn superoxide dismutase and thioredoxin and chaperones such as α‐hemoglobin stabilizing protein and HSP‐70 were upregulated in HbEβ‐thalassemia. We have also observed larger amounts of membrane associated globin chains and indications of disruption of spectrin‐based junctional complex in the membrane skeleton of HbEβ‐thalassemic erythrocytes upon detection of low molecular weight fragments of β‐spectrin and decrease in β‐actin and dematin content. Conclusion and clinical relevance: We have observed interesting changes in the proteomic levels of redox regulators and chaperons in the thalassemic hemolysates and have observed strong correlation or association of the extent of such proteomic changes with HbE levels. This could be important in understanding the role of HbE in disease progression and pathophysiology.     

Vascular proteomics

The characterization of patients with acute coronary syndromes (ACS) at the molecular and cellular levels provides a novel vision for understanding the pathological and clinical expression of the disease. Recent advances in proteomic technologies permit the evaluation of systematic changes in protein expression in many biological systems and have been extensively applied to cardiovascular diseases (CVD). The cardiovascular system is in permanent intimate contact with blood, making blood-based biomarker discovery a particularly worthwhile approach. Thus, proteomics can potentially yield novel biomarkers reflecting CVD, establish earlier detection strategies, and monitor response to therapy. Here we review the different proteomic strategies used in the study of atherosclerosis and the novel proteins differentially expressed and secreted by atherosclerotic lesions which constitute novel potential biomarkers (HSP-27, Cathepsin D). Special attention is paid to MS-Imaging of atheroma plaque and the generation, for the first time, of 2-D images of lipids, showing the distribution of these molecules in the different areas of the atherosclerotic lesions. In addition new potential biomarkers have been identified in plasma (amyloid A1α, transtherytin), circulating cells (protein profile in monocytes from ACS patients) and individual cells constituents of atheroma plaques (endothelial, VSMC, macrophages) which provide novel insights into vascular pathophysiology.     

Using proteomics to uncover extracellular matrix interactions during cardiac remodeling

The left ventricle (LV) responds to a myocardial infarction with an orchestrated sequence of events that result in fundamental changes to both the structure and function of the myocardium. This collection of responses is termed as LV remodeling. Myocardial ischemia resulting in necrosis is the initiating event that culminates in the formation of an extracellular matrix (ECM) rich infarct scar that replaces necrotic myocytes. While the cardiomyocyte is the major cell type that responds to ischemia, infiltrating leukocytes and cardiac fibroblasts coordinate the subsequent wound healing response. The matrix metalloproteinase family of enzymes regulates the inflammatory and ECM responses that modulate scar formation. Matridomics is the proteomic evaluation focused on ECM, while degradomics is the proteomic evaluation of proteases as well as their inhibitors and substrates. This review will summarize the use of proteomics to better understand matrix metalloproteinase roles in post myocardial infarction LV remodeling.     

A modeling study predicts the presence of voltage gated Ca2+ channels on myelinated central axons

The objective of this current study was to investigate whether voltage gated Ca(2+) channels are present on axons of the adult rat optic nerve (RON). Simulations of axonal excitability using a Hodgkin-Huxley based one-compartment model incorporating I(Na), I(K) and leak currents were used to predict conditions under which the potential contribution of a Ca(2+) current to an evoked action potential could be measured. Under control conditions the inclusion of a high threshold Ca(2+) current (I(Ca)) in the model had a negligible effect on the action potential. Reducing I(K), by decreasing the value of g(K), elongated the repolarizing phase of the action potential, increasing its duration. Subsequent incorporation of I(Ca) in the model revealed a significant I(Ca) contribution to the repolarizing phase of the action potential. The simulation thus suggests that Ca(2+) channels may be present on RON axons, but that pharmacological intervention is required to unmask their presence. Experiments based on the simulations revealed that there was no significant contribution of I(Ca) to the control action potential. However, as predicted by the simulation, reducing the repolarizing effect of I(K) by adding the K(+) channel blocker 4-AP revealed a Ca(2+) component on the repolarizing phase of the action potential that was blocked by the Ca(2+) channel inhibitor nifedipine.     

Quantitative proteomic approaches for biomarker discovery

The rapid advances in proteomic technologies have made possible systematic analysis of hundreds to thousands of proteins in clinical samples with the promise of uncovering novel protein biomarkers for various disease conditions. We will discuss in this review article current MS and protein chip-based quantitative proteomic approaches and their application in biomarker discovery. The emphasis will be placed on new quantification strategies employing stable isotopic labeling coupled with MS/MS, and antibody-based protein chips and nanodevices. The strength and weakness of each technology are briefly highlighted.     

Quantitative mass spectrometry for colorectal cancer proteomics

This review documents the uses of quantitative MS applied to colorectal cancer (CRC) proteomics for biomarker discovery and molecular pathway profiling. Investigators are adopting various labeling and label-free MS approaches to quantitate differential protein levels in cells, tumors, and plasma/serum. We comprehensively review recent uses of this technology to examine mouse models of CRC, CRC cell lines, their secretomes and subcellular fractions, CRC tumors, CRC patient plasma/serum, and stool samples. For biomarker discovery these approaches are uncovering proteins with potential diagnostic and prognostic utility, while in vitro cell culture experiments are characterizing proteomic and phosphoproteomic responses to disrupted signaling pathways due to mutations or to inhibition of drugable enzymes.     

Human pathogenic streptococcal proteomics and vaccine development

Gram-positive streptococci are non-motile, chain-forming bacteria commonly found in the normal oral and bowel flora of warm-blooded animals. Over the past decade, a proteomic approach combining 2-DE and MS has been used to systematically map the cellular, surface-associated and secreted proteins of human pathogenic streptococcal species. The public availability of complete streptococcal genomic sequences and the amalgamation of proteomic, genomic and bioinformatic technologies have recently facilitated the identification of novel streptococcal vaccine candidate antigens and therapeutic agents. The objective of this review is to examine the constituents of the streptococcal cell wall and secreted proteome, the mechanisms of transport of surface and secreted proteins, and describe the current methodologies employed for the identification of novel surface-displayed proteins and potential vaccine antigens.