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6. Site-specific quantitation of protein nitration using liquid chromatography/tandem mass spectrometry

Author

Willard, Belinda B., Ruse, Cristian I., Keightley, Andrew J., Bond, Meredith, and Kinter, Michael

Subjects
Chemistry, Analytic -- Research, Mass spectrometry -- Usage, Chemistry
Abstract

The native reference peptide (NRP) method has been adapted to the measure of the degree of protein nitration at a specific tyrosine residue. In these experiments, human serum albumin was modified in a myeloperoxidase-mediated reaction in the presence of nitrite, with nitration detected predominantly at one site, [Y.sup.162]. The time-dependent increase in nitration at this site was measured based on the increasing abundance of the peptide [sup.162][Y.sup.n]LYEIA[R.sup.168] and the corresponding decrease in the [sup.162]YLYEIA[R.sup.168] peptide in in-gel trypsin digests. The peptide [sup.66]LVNEVIEFA[K.sup.75], also formed in the tryptic digest, was used as the native reference peptide. Quantitation was achieved by determining the chromatographic peak area of the two analyte peptides relative to the native reference peptide by LC/tandem mass spectrometric analyses with selected reaction monitoring. The NRP results were validated by correlation to the time-dependent increase in total protein-nitrotyrosine content determined by Western blot analysis. The precision and limit of detection of the assay were also evaluated and were found to be approximately 10% (relative standard deviation) and 5 fmol on-column, respectively. These results demonstrate the utility of the NRP method for quantitative analyses of posttranslation modifications, in terms of broad applicability, ease of experimental design, sensitivity, and precision.

Published
2003

7. Quantitative dynamics of site-specific protein phosphorylation determined using liquid chromatography electrospray ionization mass spectrometry

Author

Ruse, Cristian I., Willard, Belinda, Jin, J.P., Haas, Thomas, Kinter, Michael, and Bond, Meredith

Subjects
Secondary ion mass spectrometry -- Methods, Liquid chromatography -- Methods, Phosphorylation -- Measurement, Proteins -- Analysis, Chemistry
Abstract

We have developed and validated a method that uses liquid chromatography/electrospray ionization-mass spectrometry to quantify site-specific protein phosphorylation. The method uses selected ion monitoring to determine the chromatographic peak areas of specific tryptic peptides from the protein of interest. The extent of phosphorylation is determined from the ratio of the phosphopeptide peak area to the peak area of an unmodified reference peptide that acts as internal standard, correcting for variations in protein amounts and peptide recovery in the digest preparation procedure. As a result, we refer to this protocol as the native reference peptide method. Mole of phosphate at the selected site per mole of protein is obtained from this ratio, using calibration curves of synthetic peptides to determine relative responses. Our method begins with protein separation by SDS-PAGE and is carried out on amounts of peptide produced by an ingel digestion of single Coomassie blue-stained bands. To illustrate the utility of the method and provide validation, we used cardiac troponin I as analyte and monitored the time course of a protein kinase C [beta]II reaction. Those analyses appropriately demonstrate the time-dependent increase of phosphorylation at a PKC-preferred site, Ser44 in the peptide [sup.41][ISAS.sup.p][R.sup.45] and the concomitant consumption of the nonphosphorylated peptide. We believe that this method provides a novel tool to directly measure specific phosphorylation sites in proteins in different physiological states and expect that the method will be adaptable not only to a variety of samples types (i.e., culture cells, tissues, etc.) but to a variety of posttranslation modifications as well.

Published
2002

8. The highly conserved COOH terminus of troponin I forms a Ca(super 2+)-modulated allosteric domain in the troponin complex

Author

Jin, Jian-Ping, Yang, Fang-Wei, Yu, Zhi-Bin, Ruse, Cristian I., Bond, Meredith, and Chen, Aihua

Subjects
Muscle proteins -- Research, Proteins -- Structure, Biological sciences, Chemistry
Abstract

The COOH terminus of troponin I is essential in the binding of troponin C, T, and I in the presence of calcium. The COOH terminus is exposed in the muscle thin filament and participates in the allosteric switch during muscle contraction.

Published
2001

9. Troponin I phosphorylation and myofilament calcium sensitivity during decompensated cardiac hypertrophy

Author

McConnell, Bradley K., Moravec, Christine Schomisch, and Bond, Meredith

Subjects
Heart cells -- Physiological aspects, Heart enlargement -- Physiological aspects, Heart failure -- Physiological aspects, Biological sciences
Abstract

Spontaneously hypertensive rats (SHR) are used to evaluate the myofilament calcium sensitivity-associated alteration in troponin-1 (Tn-1) phosphorylation and the expression of troponin isoforms during the evolution to decompensated cardiac hypertrophy. Results indicate that Tn-1 phosphorylation upon beta adrenergic stimulation of myocytes decreased among SHRs. The implications of these findings are discussed.

Published
1998

10. Troponin I phosphorylation in spontaneously hypertensive rat heart: effect of beta-adrenergic stimulation

Author

McConnell, Bradley K., Moravec, Christine Schomisch, Morano, Ingo, and Bond, Meredith

Subjects
Protein kinases -- Physiological aspects, Phosphorylation -- Physiological aspects, Heart cells -- Physiological aspects, Biological sciences
Abstract

A study was performed to compare the baseline and protein kinase A (PKA)-dependent troponin I (TnI) phosphorylation in left ventricular myocytes from hearts of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Phosphorylation of TnI by PKA was believed to reduce myofilament Ca2+ sensitivity. Results indicated that after activation of the beta-adrenergic mechanism, PKA-dependent TnI phosphorylation from the SHR group was greater than in WKY coupled with decreased Ca2+ sensitivity of actomyosin ATPase activity.

Published
1997

11. Mitochondrial calcium content in isolated perfused heart: effects of inotropic stimulation

Author

Moravec, Christine Schomisch, Desnoyer, Russell W., Milovanovic, Milorad, Schluchter, Mark D., and Bond, Meredith

Subjects
Mitochondria -- Physiological aspects, Calcium in the body -- Physiological aspects, Biological sciences
Abstract

A study was initiated to determine effects of mitochondrial metabolism on mitochondrial calcium (Ca) uptake during physiological processes. Mensuration of changes in the left ventricular function, pyruvate dehydrogenase (PDH) activity and mitochondrial Ca content was done in perfused hamster hearts frozen during systole. Results indicated that no relevant change was seen in mitchondrial Ca content during isoproterenol activation and increased perfusion pressure.

Published
1997

14. Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice

Author

McConnell, Bradley K., Jones, Karen A., Fatkin, Diane, Arroyo, Luis H., Lee, Richard T., Aristizabal, Orlando, Turnbull, Daniel H., Georgakopoulos, Dimitrios, Kass, David, Bond, Meredith, Niimura, Hideshi, Schoen, Frederick J., Conner, David, Fischman, Donald H., Seidman, Christine E., and Seidman, J. G.

Published
1999

20. Disruption of Protein Kinase A Interaction with A-kinase-anchoring Proteins in the Heart in Vivo: EFFECTS ON CARDIAC CONTRACTILITY, PROTEIN KINASE A PHOSPHORYLATION, AND TROPONIN I PROTEOLYSIS*

Author

McConnell, Bradley K., Popovic, Zoran, Mal, Niladri, Lee, Kwangdeok, Bautista, James, Forudi, Farhad, Schwartzman, Raul, Jin, J.-P., Penn, Marc, and Bond, Meredith

Subjects
Male, Cardiotonic Agents, Myocardium, Mechanisms of Signal Transduction, Calcium-Binding Proteins, Troponin I, Isoproterenol, A Kinase Anchor Proteins, Gene Expression, Cyclic AMP-Dependent Protein Kinase Type II, Ryanodine Receptor Calcium Release Channel, Stroke Volume, macromolecular substances, musculoskeletal system, Myocardial Contraction, Adenoviridae, Rats, Mice, Transduction, Genetic, cardiovascular system, Animals, Phosphorylation, Peptides
Abstract

Protein kinase A (PKA)-dependent phosphorylation is regulated by targeting of PKA to its substrate as a result of binding of regulatory subunit, R, to A-kinase-anchoring proteins (AKAPs). We investigated the effects of disrupting PKA targeting to AKAPs in the heart by expressing the 24-amino acid regulatory subunit RII-binding peptide, Ht31, its inactive analog, Ht31P, or enhanced green fluorescent protein by adenoviral gene transfer into rat hearts in vivo. Ht31 expression resulted in loss of the striated staining pattern of type II PKA (RII), indicating loss of PKA from binding sites on endogenous AKAPs. In the absence of isoproterenol stimulation, Ht31-expressing hearts had decreased +dP/dtmax and -dP/dtmin but no change in left ventricular ejection fraction or stroke volume and decreased end diastolic pressure versus controls. This suggests that cardiac output is unchanged despite decreased +dP/dt and -dP/dt. There was also no difference in PKA phosphorylation of cardiac troponin I (cTnI), phospholamban, or ryanodine receptor (RyR2). Upon isoproterenol infusion, +dP/dtmax and -dP/dtmin did not differ between Ht31 hearts and controls. At higher doses of isoproterenol, left ventricular ejection fraction and stroke volume increased versus isoproterenol-stimulated controls. This occurred in the context of decreased PKA phosphorylation of cTnI, RyR2, and phospholamban versus controls. We previously showed that expression of N-terminal-cleaved cTnI (cTnI-ND) in transgenic mice improves cardiac function. Increased cTnI N-terminal truncation was also observed in Ht31-expressing hearts versus controls. Increased cTnI-ND may help compensate for reduced PKA phosphorylation as occurs in heart failure.

Published
2009

23. Characterization of Skeletal Muscle in the Synemin Knock-out Mouse.

Author

García-Pelagio, Karla P., Muriel, Joaquin, Lovering, Richard M., Lund, Linda, Bond, Meredith, and Bloch, Robert J.

Subjects
SKELETAL muscle, SYNEMIN, COSTAMERES, CONTRACTILE proteins, CELL membranes, MESSENGER RNA, LABORATORY mice, DISEASES
Abstract

Diseases linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus of skeletal and cardiac muscle and its links to costameres, which connect the sarcomeres to the cell membrane. Synemin is a large IF protein that associates with dystrobrevin, vinculin, and talin at costameres of the cell membrane of striated muscle, as well as with a-actinin and desmin at the Z disks. Synemin can be expressed in either 210 kDa a- or 180 kDa ß- alternatively spliced forms. We generated mice null for synemin by homologous recombination to study synemin's function in skeletal muscle. Skeletal muscle in the knock out (syn KO) mouse does not make synemin mRNA or protein. Preliminary characterization of the syn KO mouse suggests that it has a mild skeletal muscle phenotype. The organization of costameres appears to be normal. Treadmill running uphill test results was not significantly affected when compared to controls at any age. More notably, the biomechanical properties of the cell membrane are different in the syn KO, though they are less affected than by the absence of desmin or dystrophin. These results suggest that the viscoelastic properties of the cell membrane-costamere-myofibril complex are significantly influenced by synemin. [ABSTRACT FROM AUTHOR]

Published
2014
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28. Measurement of Subcellular CA2+ Redistribution in Cardiac Muscle In Situ: Time Resolved Rapid Freezing and Electron Probe Microanalysis

Author

Bond, Meredith, Schluchter, Mark D., Keller, Eva, and Moravec, Christine S.

Subjects
mitochondria, cryosection, analysis of variance, calcium, Electron probe microanalysis, heart, papillary muscle, contractile force, junctional sarcoplasmic reticulum, Biology, time-resolved rapid freezing
Abstract

To directly assess the physiological roles of sarcoplasmic reticulum (SR) and miitochondria (MT), we have utilized energy dispersive electron probe microanalysis (EPMA) on ultrathin freeze-dried cryosections from isolated papillary muscles, rapidly frozen at precise time points of the contractile cycle. Using this approach, we can detect redistribution of subcellular Ca2+ during the cardiac contractile cycle. Changes in Ca2+ of less than 1.0 mmol/kg dry wt can be detected. By determining the variability of the Ca2+ measurements in preliminary experiments, we have also demonstrated that it is possible to optimize experimental design, i.e., to predict the number of animals per treatment group and the number of X-ray spectra per animal that are required in order to detect a specified Ca2+ difference. Quantitative EPMA of rapidly frozen contracting papillary muscle has also allowed us to correlate the Ca2+ content of SR and MT with the contractile state of the muscle. Our results show a decrease of 40% in the amount of Ca2+ stored in the junctional SR during a cardiac muscle twitch, thus providing direct evidence for a role of the SR as a primary site of Ca2+ release. In addition, we have demonstrated dissociation between MT Ca2+ uptake and activation of regulatory enzymes, such as pyruvate dehydrogenase, indicating that MT Ca2+ uptake is not required for activation of MT metabolism.

Published
1994

39. Sustained apoptosis in human cardiac allografts despite histologic resolution of rejection1

Author

Masri, Sofia C., primary, Yamani, Mohamad H., additional, Russell, Mary A., additional, Ratliff, Norman B., additional, Yang, Jiacheng, additional, Almasan, Alex, additional, Apperson-Hansen, Carolyn, additional, Li, Jianbo, additional, Starling, Randall C., additional, McCarthy, Patrick, additional, Young, James B., additional, and Bond, Meredith, additional

Published
2003
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48. Decreased SLIM1 Expression and Increased Gelsolin Expression in Failing Human Hearts Measured by High-Density Oligonucleotide Arrays

Author

Yang, Jiacheng, primary, Moravec, Christine S., additional, Sussman, Mark A., additional, DiPaola, Nicholas R., additional, Fu, Dechen, additional, Hawthorn, Lesley, additional, Mitchell, Christina A., additional, Young, James B., additional, Francis, Gary S., additional, McCarthy, Patrick M., additional, and Bond, Meredith, additional

Published
2000
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