Cardiac myosin-binding protein C: a potential early biomarker of myocardial injury.

Cardiac troponins are released and cleared slowly after myocardial injury, complicating the diagnosis of early, and recurrent, acute myocardial infarction. Cardiac myosin-binding protein C (cMyC) is a similarly cardiac-restricted protein that may have different release/clearance kinetics. Using novel antibodies raised against the cardiac-specific N-terminus of cMyC, we used confocal microscopy, immunoblotting and immunoassay to document its location and release. In rodents, we demonstrate rapid release of cMyC using in vitro and in vivo models of acute myocardial infarction. In patients, with ST elevation myocardial infarction (STEMI, n = 20), undergoing therapeutic ablation of septal hypertrophy (TASH, n = 20) or having coronary artery bypass surgery (CABG, n = 20), serum was collected prospectively and frequently. cMyC appears in the serum as full-length and fragmented protein. Compared to cTnT measured using a contemporary high-sensitivity commercial assay, cMyC peaks earlier (STEMI, 9.3 ± 3.1 vs 11.8 ± 3.4 h, P < 0.007; TASH, 9.7 ± 1.4 vs 21.6 ± 1.4 h, P < 0.0001), accumulates more rapidly (during first 4 h after TASH, 25.8 ± 1.9 vs 4.0 ± 0.4 ng/L/min, P < 0.0001) and disappears more rapidly (post-CABG, decay half-time 5.5 ± 0.8 vs 22 ± 5 h, P < 0.0001). Our results demonstrate that following defined myocardial injury, the rise and fall in the serum of cMyC is more rapid than that of cTnT. We speculate that these characteristics could enable earlier diagnosis of myocardial infarction and reinfarction in suspected non-STEMI, a population not included in this early translational study.

Redox proteomics in study of kidney-associated hypertension: new insights to old diseases.

SIGNIFICANCE: The kidney helps to maintain low blood pressure in the human body, and impaired kidney function is a common attribute of aging that is often associated with high blood pressure (hypertension). Kidney-related pathologies are important contributors (either directly or indirectly) to overall human mortality. In comparison with other organs, kidney has an unusually wide range of oxidative status, ranging from the well-perfused cortex to near-anoxic medulla. RECENT ADVANCES: Oxidative stress has been implicated in many kidney pathologies, especially chronic kidney disease, and there is considerable research interest in oxidative stress biomarkers for earlier prediction of disease onset. Proteomics approaches have been taken to study of human kidney tissue, serum/plasma, urine, and animal models of hypertension. CRITICAL ISSUES: Redox proteomics, in which oxidative post-translational modifications can be identified in protein targets of oxidative or nitrosative stress, has not been very extensively pursued in this set of pathologies. FUTURE DIRECTIONS: Proteomics studies of kidney and related tissues have relevance to chronic kidney disease, and redox proteomics, in particular, represents an under-exploited toolkit for identification of novel biomarkers in this commonly occurring pathology.

Proteomics in investigation of protein nitration in kidney disease: technical challenges and perspectives from the spontaneously hypertensive rat.

Kidneys are the mammalian organs with widest range of oxidative status ranging from the well-perfused cortex to the relatively anoxic medulla. This organ is of key interest from the perspective of hypertension, an important contributor to human mortality, and there has been growing use of the spontaneously hypertensive rat (SHR) as a model to explore oxidative stress in hypertensive kidney. Nitrosative stress is often associated with oxidative stress and, like oxidative stress, can lead to covalent modification of protein side-chains. It is especially relevant to kidney because of high levels of both nitrite/nitrate and nitric oxide synthase in medulla. Because of their relatively low abundance and their well-known role in signal transduction, nitration of tyrosines to 3-nitrotyrosines (3NT) is of particular interest in this regard. This modification has the potential to contribute to changes in regulation, in protein activity and may provide a means of specific targeting of key proteins. Mass spectrometry (MS) offers a promising route to detecting this modification. This review surveys protein nitration in kidney disease and highlights opportunities for MS detection of nitrated residues in the SHR.

Proteomic profiling of perturbed protein sulfenation in renal medulla of the spontaneously hypertensive rat.

Protein sulfenic acids have been proposed as potential biochemical switches for redox signaling. This post-translational modification (PTM) is readily reversible, in contrast to some other types of oxidative PTM. Enhanced oxidative stress has been reported as a feature of hypertension, and renal function has been implicated in the development and progression of the disease in animal models such as the spontaneously hypertensive rat (SHR). However, reactive oxygen species (ROS) are also signaling molecules and may play a role in vascular function. To investigate protein sulfenation under hypertensive conditions, we examined protein extracts of SHR kidney medulla in comparison to medulla from normotensive Wistar rats. Total free thiol content of the SHR medulla was significantly lower than that of Wistar medulla, indicating enhanced oxidation of sulfhydryls. Protein sulfenation was also significantly greater in the medulla of hypertensive animals. Thioredoxin reductase activity was also reduced in SHR medulla and this may account, in part, for enhanced protein sulfenation. Purification of sulfenated proteins from SHR medulla revealed several proteins involved in processes such as metabolism, antioxidant defense, and regulation of nitric oxide synthase. Enhanced sulfenation may represent perturbed redox signaling in SHR medulla, or simply enhanced ROS generation.

Strategies for analysing and improving the expression and quality of recombinant proteins made in mammalian cells.

The production of monoclonal antibodies and other recombinant proteins is one of the highest growth areas in the pharmaceutical industry. Mammalian cells are used to manufacture the majority of biotherapeutics, largely due to their ability to perform complex post-translational modifications. Although significant progress has been made recently in improving product yields and protein quality, many challenges still lie ahead to achieve consistently high yields while avoiding potentially damaging protein modifications. The present review first considers the strategies used to analyse and improve recombinant protein expression of industrial cell lines, with an emphasis on proteomic technologies. Next, cellular and environmental influences on protein production and quality are examined, and strategies for improvements in product yield and quality are reviewed. The analytical techniques required to detect these protein changes are also described, together with prospects for assay improvements.

Protein carbonylation in kidney medulla of the spontaneously hypertensive rat.

Enhanced generation of ROS has been reported in models of hypertension such as the spontaneously hypertensive rat (SHR). Impairment of kidney function has been implicated in development and progression of hypertension, and the renal medulla appears to play an important role in regulating long-term blood pressure. A key biomarker of oxidative stress is the formation of protein carbonyls, which we set out to characterize in the SHR medulla. We identified 11 proteins that were differentially carbonylated in SHR medulla in comparison to normotensive wistars including enolase 1, catalase, carbonic anhydrase II, transferrin and members of the aldo-keto-reductase family. This enhanced protein oxidation was not only accompanied by an increase in intracellular iron deposition, but aldo-keto-reductase activity was also significantly less in SHR medulla than in normotensive Wistars. Oxidative stress appears selectively to target a subset of proteins in SHR kidney and modification of these proteins may in turn contribute to the renopathy associated with hypertension.

Proteomic identification of tyrosine nitration targets in kidney of spontaneously hypertensive rats.

Nitrosative and oxidative stress are implicated in the development of hypertension. Events in the renal medulla may play a key role in the development and progression of hypertension. This may arise through disruption of nitric oxide signalling in the medulla and be accompanied by enhanced nitrosative and oxidative stress as indicated by the presence of proteins containing 3-nitrotyrosine. Here we demonstrate enhanced protein nitration in the medulla of spontaneously hypertensive rats. We have identified several nitrated proteins with both varied subcellular location and functional roles. These proteins are involved in nitric oxide signalling, antioxidant defense and energy metabolism. Moreover, increased nitration was observed in conjunction with enhanced oxidative damage as evidenced by the presence of protein carbonyl oxidative stress biomarkers. Our results suggest that kidney medulla is subject to enhanced nitrosative and oxidative stress, and that resulting protein modifications may contribute to the progression of hypertension.

Carbonylation and glutathionylation of proteins in the blue mussel Mytilus edulis detected by proteomic analysis and Western blotting: Actin as a target for oxidative stress.

Protein expression profiles (PEPs) were generated by two-dimensional electrophoresis (2-D SDS-PAGE) for gill and digestive glands of Mytilus edulis sampled from a polluted and reference site in Cork Harbour, Ireland. Similar patterns and expression levels were found for both sites in silver stained gels. However, Western blotting for carbonylated proteins demonstrated higher levels of specific carbonylation of proteins in tissues from animals in the polluted site. Animals from the reference site were acclimated in holding tanks, exposed to 1 mM H2O2 for 24 h, dissected and analysed by 2-D SDS-PAGE. Again, generally similar PEPs were found in control and exposed animals for gill and digestive gland but carbonylation was more pronounced in polluted and exposed animals. Western blotting of extracts after one-dimensional electrophoresis with antibodies to glutathione and actin revealed that gill proteins are glutathionylated more strongly than digestive gland and that this process is more pronounced in polluted animals than in controls. We conclude that carbonylation and glutathionylation can occur in gill and digestive gland in response to oxidative stress in M. edulis. Actin is a major target for both glutathionylation and carbonylation under oxidative stress conditions.