Initial analysis of the phosphoproteome of Chinese hamster ovary cells using electrophoresis.
AuthorsChen Z, Southwick K, Thulin CD
JournalJ Biomol Tech
PubMed ID15585821
'Protein phosphorylation is a common post-translational modification of enormous biological importance. Analysis of phosphorylation at the global level should shed light on the use of this modification to regulate metabolism, signal transduction, and other processes. We have begun a proteomic analysis of phosphorylation using two-dimensional gel electrophoresis. Chinese hamster ovary ... More
Comparative proteomes of the proliferating C(2)C(12) myoblasts and fully differentiated myotubes reveal the complexity of the skeletal muscle differentiation program.
AuthorsTannu NS, Rao VK, Chaudhary RM, Giorgianni F, Saeed AE, Gao Y, Raghow R
JournalMol Cell Proteomics
PubMed ID15286212
'When cultured in low serum-containing growth medium, the mouse C(2)C(12) cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and ... More
Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry.
'Protein phosphorylation plays a vital role in the regulation of most aspects of cellular activity, being key to propagating messages within signal transduction pathways and to modulating protein function. Pro-Q Diamond phosphoprotein gel stain is suitable for the fluorescence detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl ... More
Chronophin, a novel HAD-type serine protein phosphatase, regulates cofilin-dependent actin dynamics.
AuthorsGohla A, Birkenfeld J, Bokoch GM
JournalNat Cell Biol
PubMed ID15580268
Cofilin is a key regulator of actin cytoskeletal dynamics whose activity is controlled by phosphorylation of a single serine residue. We report the biochemical isolation of chronophin (CIN), a unique cofilin-activating phosphatase of the haloacid dehalogenase (HAD) superfamily. CIN directly dephosphorylates cofilin with high specificity and colocalizes with cofilin in ... More
Determination of in vivo protein phosphorylation in photosynthetic membranes.
AuthorsVainonen JP, Vener AV, Aro EM,
JournalMethods Mol Biol
PubMed ID19083170
Light- and redox-controlled reversible phosphorylation of thylakoid proteins regulates short- and long-term acclimation of plants to environmental cues. The major phosphoproteins in thylakoids belong to photosystem II and its light-harvesting antenna but phosphorylation of subunits of other thylakoid protein complexes has been detected as well. The detection methods include electrophoretic ... More
A high-resolution two dimensional Gel- and Pro-Q DPS-based proteomics workflow for phosphoprotein identification and quantitative profiling.
AuthorsAgrawal GK, Thelen JJ,
JournalMethods Mol Biol
PubMed ID19241001
The two-dimensional (2-D) gel-based proteomics platform remains the workhorse for proteomics and is fueled by a number of key improvements, including fluorescence-based stains for detection and quantification of proteins and phosphoproteins with high sensitivity and linear dynamic ranges. One such stain is Pro-Q diamond phosphoprotein stain (Pro-Q DPS), which binds ... More
Detection of post-translational modifications by fluorescent staining of two-dimensional gels.
AuthorsJacob AM, Turck CW,
JournalMethods Mol Biol
PubMed ID18373247
Post-translational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial in the discovery of biomarkers of disease. Recent commercial availability of fluorescent dyes specifically staining PTMs of proteins such as phosphorylation and glycosylation enables the specific ... More
Structural and Functional Characterization of NikO, an Enolpyruvyl Transferase Essential in Nikkomycin Biosynthesis.
AuthorsOberdorfer G, Binter A, Ginj C, Macheroux P, Gruber K,
JournalJ Biol Chem
PubMed ID22810238
Nikkomycins are peptide-nucleoside compounds with fungicidal, acaricidal, and insecticidal properties because of their strong inhibition of chitin synthase. Thus, they are potential antibiotics especially for the treatment of immunosuppressed patients, for those undergoing chemotherapy, or after organ transplants. Although their chemical structure has been known for more than 30 years, ... More
Protein gel staining methods: an introduction and overview.
AuthorsSteinberg TH,
JournalMethods Enzymol
PubMed ID19892191
Laboratory scientists who encounter protein biochemistry in many of its myriad forms must often ask: is my protein pure? The most frequent response: run a denaturing SDS polyacrylamide gel. Running this gel raises another series of considerations regarding detection, quantitation, and characterization and so the next questions invariably center on ... More
Increased phosphorylation of tropomyosin, troponin I, and myosin light chain-2 after stretch in rabbit ventricular myocardium under physiological conditions.
AuthorsMonasky MM, Biesiadecki BJ, Janssen PM,
JournalJ Mol Cell Cardiol
PubMed ID20298699
After a change in muscle length, there is an immediate intrinsic response in the amount of developed force, followed by a slower response. Although it has been well documented that the slow force response is at least in part generated by modification of calcium handling, it is unclear whether regulation ... More
Use of a fluorescent phosphoprotein dye to characterize oxidative stress-induced signaling pathway components in macrophage and epithelial cultures exposed to diesel exhaust particle chemicals.
AuthorsWang M, Xiao GG, Li N, Xie Y, Loo JA, Nel AE
JournalElectrophoresis
PubMed ID15880549
A large body of evidence has shown that exposure to ambient particulate matter (PM) leads to asthma exacerbation through an excitation of allergic inflammation. Utilizing diesel exhaust particles (DEPs) as a model air pollutant, we and others have demonstrated that PM contains redox-active chemicals that generate inflammation through an oxidative ... More
Focused proteomics: monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain.
AuthorsMurray J, Marusich MF, Capaldi RA, Aggeler R
JournalElectrophoresis
PubMed ID15300771
We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained ... More
Involvement of RhoA-mediated Ca2+ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice.
AuthorsChiba Y, Ueno A, Shinozaki K, Takeyama H, Nakazawa S, Sakai H, Misawa M
JournalRespir Res
PubMed ID15638941
BACKGROUND: It has recently been suggested that RhoA plays an important role in the enhancement of the Ca2+ sensitization of smooth muscle contraction. In the present study, a participation of RhoA-mediated Ca2+ sensitization in the augmented bronchial smooth muscle (BSM) contraction in a murine model of allergic asthma was examined. ... More
Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye.
The phosphorylation of mitochondrial proteins is pivotal to the regulation of respiratory activity in the cell and to signaling pathways leading to apoptosis, as well as for other vital mitochondrial processes. A number of protein kinases have been identified in mitochondria but the physiological substrates for many of these remain ... More
Functional effects of rho-kinase-dependent phosphorylation of specific sites on cardiac troponin.
AuthorsVahebi S, Kobayashi T, Warren CM, de Tombe PP, Solaro RJ
JournalCirc Res
PubMed ID15774859
We tested the hypothesis that activation of Rho-A-dependent kinase (ROCK-II) alters cardiac myofilament response to Ca2+ by mechanisms involving phosphorylation of thin filament proteins. We determined effects of a constitutively active form of ROCK-II on ATPase activity and tension development in detergent-extracted (skinned) fiber bundles isolated from mouse left ventricular ... More
Proteomic analysis of in vivo phosphorylated synaptic proteins.
AuthorsCollins MO, Yu L, Coba MP, Husi H, Campuzano I, Blackstock WP, Choudhary JS, Grant SG
JournalJ Biol Chem
PubMed ID15572359
In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue ... More
Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase.
AuthorsWang X, Goshe MB, Soderblom EJ, Phinney BS, Kuchar JA, Li J, Asami T, Yoshida S, Huber SC, Clouse SD
JournalPlant Cell
PubMed ID15894717
Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in ... More
Functional analyses of placental protein 13/galectin-13.
AuthorsThan NG, Pick E, Bellyei S, Szigeti A, Burger O, Berente Z, Janaky T, Boronkai A, Kliman H, Meiri H, Bohn H, Than GN, Sumegi B
JournalEur J Biochem
PubMed ID15009185
Placental protein 13 (PP13) was cloned from human term placenta. As sequence analyses, alignments and computational modelling showed its conserved structural and functional homology to members of the galectin family, the protein was designated galectin-13. Similar to human eosinophil Charcot-Leyden crystal protein/galectin-10 but not other galectins, its weak lysophospholipase activity ... More
Functional characterization of two-dimensional gel-separated proteins using sequential staining.
Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications ... More
Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis.
AuthorsAllanore Y, Borderie D, Périanin A, Lemaréchal H, Ekindjian OG, Kahan A
JournalArthritis Res Ther
PubMed ID15642147
We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects in vivo and in vitro of nifedipine on superoxide anion (O2*-) production by peripheral ... More
Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye.
Protein kinases represent one of the largest families of regulatory enzymes, with more than 2,000 of them being encoded for by the human genome. Many cellular processes are regulated by the reversible phosphorylation of proteins and upwards of 30% of the proteins comprising the eukaryotic proteome are likely to be ... More
Use of proteomics to demonstrate a hierarchical oxidative stress response to diesel exhaust particle chemicals in a macrophage cell line.
AuthorsXiao GG, Wang M, Li N, Loo JA, Nel AE
JournalJ Biol Chem
PubMed ID14522998
Epidemiological studies demonstrate an association between short term exposure to ambient particulate matter (PM) and cardiorespiratory morbidity and mortality. Although the biological mechanisms of these adverse effects are unknown, emerging data suggest a key role for oxidative stress. Ambient PM and diesel exhaust particles (DEP) contain redox cycling organic chemicals ... More
Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye.
Ultrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post-translational modifications, such as phosphorylation. However, the analysis of ... More
Detection of phosphoproteins on electroblot membranes using a small-molecule organic fluorophore.
A new formulation of the small-molecule organic fluorophore, Pro-Q Diamond dye, has been developed that permits rapid and simple detection of phosphoproteins directly on polyvinylidene difluoride (PVDF) or nitrocellulose membranes (electroblots). Protein samples are first separated by electrophoresis and then electroblotted to membranes, stained and destained, in an analogous manner ... More