We have developed a monoclonal antibody that specifically interacts with a surface antigen of hum... more We have developed a monoclonal antibody that specifically interacts with a surface antigen of human fibroblasts and smooth muscle cells. The antibody (antibody IIGlO) recognizes a polypeptide of molecular mass 330,000, revealed by immunoblotting in fibroblast and smooth muscle cell extract, but not in vascular endothelial cells, peritoneal macrophages, peripheral blood lymphocytes nor hepatocytes. In tissue sections the antibody stained smooth muscle cells of myometrium, aorta and smaller blood vessels, and fibroblasts of connective tissue. Specificity of the antibody was further confirmed by double staining of aorta sections. Antibody IIGlO was used to identify smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta.
Molecular chaperones and cytosolic stress proteins are actively involved in the stabilization, im... more Molecular chaperones and cytosolic stress proteins are actively involved in the stabilization, import and refolding of precursor proteins into mitochondria. The purpose of the present study was to evaluate the relationship between mitochondrial content under steady-state conditions, and during the induction of organelle biogenesis, with the expression of stress proteins and mitochondrial chaperonins. A comparison of steady-state levels of mitochondrial enzyme activity [cytochrome c oxidase (CYTOX)] with chaperonin levels [the heat-shock protein HSP60, the glucose-regulated protein GRP75 (mtHSP70)] in striated muscles possessing a wide range of oxidative capacities revealed a proportional expression between the two. This relationship was disrupted by chronic contractile activity brought about by 10 days of 10 Hz stimulation of the tibialis anterior (TA) muscle, which induced 2.4-fold increases in CYTOX activity, but 3.2- and 9.3-fold increases in HSP60 and GRP75 respectively. The ind...
The control of enzyme activity by reversible phosphorylation and the role of protein kinases in s... more The control of enzyme activity by reversible phosphorylation and the role of protein kinases in signal transduction illustrate well the pivotal role of protein phosphorylation in cell regulation. There are numerous examples of the role of protein kinases and ...
The purpose of this phospho-proteomics study was to demonstrate the broad analysis of cellular pr... more The purpose of this phospho-proteomics study was to demonstrate the broad analysis of cellular protein phosphorylation in cells and tissue as a means to monitor changes in cellular states. As a cancer model, human tumor-derived A431 cells known to express the epidermal growth factor receptor (EGFR) were grown as cell cultures or xenograft tumors in mice. The cells and tumor-bearing animals were subjected to treatments including the EGFR-directed protein kinase inhibitor PK166 and/or EGF stimulation. Whole cell/tissue protein extracts were converted to peptides by using trypsin, and phosphorylated peptides were purified by an affinity capture method. Peptides and phosphorylation sites were characterized and quantified by using a combination of tandem mass spectroscopy (MS) and Fourier transform MS instrumentation (FTMS). By analyzing roughly 10 6 cell equivalents, 780 unique phosphopeptides from approx 450 different proteins were characterized. Only a small number of these phosphorylation sites have been described previously in literature. Although a targeted analysis of the EGFR pathway was not a specific aim of this study, 22 proteins known to be associated with EGFR signaling were identified. Fifty phosphopeptides were found changed in abundance as a function of growth factor or drug treatment including novel sites of phosphorylation on the EGFR itself. These findings demonstrate the feasibility of using phospho-proteomics to determine drug and disease mechanisms, and as a measure of drug target modulation in tissue.
We have developed a monoclonal antibody that specifically interacts with a surface antigen of hum... more We have developed a monoclonal antibody that specifically interacts with a surface antigen of human fibroblasts and smooth muscle cells. The antibody (antibody IIG10) recognizes a polypeptide of molecular mass 330,000, revealed by immunoblotting in fibroblast and smooth muscle cell extract, but not in vascular endothelial cells, peritoneal macrophages, peripheral blood lymphocytes nor hepatocytes. In tissue sections the antibody stained smooth muscle cells of myometrium, aorta and smaller blood vessels, and fibroblasts of connective tissue. Specificity of the antibody was further confirmed by double staining of aorta sections. Antibody IIG10 was used to identify smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta.
Mapping protein-protein interactions is an invaluable tool for understanding protein function. He... more Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.
Dystrophin, vinculin, and aciculin in skeletal muscle subject to chronic use and disuse REZVANI, ... more Dystrophin, vinculin, and aciculin in skeletal muscle subject to chronic use and disuse REZVANI, M OJGAN; ORNATSKY, OLGA I.; CONNOR, M ICHAEL K.; EISENBERG, HERBERT A.; HOOD, DAVID A.
We describe the synthesis and characterization of element-encoded polystyrene nanoparticles with ... more We describe the synthesis and characterization of element-encoded polystyrene nanoparticles with diameters on the order of 100 nm and a narrow size distribution. Individual particles contain ca. 10 3 chelated lanthanide ions, of either a single element or a mixture of elements. These particles were effectively internalized by nonspecific endocytosis into three cell lines associated with human leukemia. Using an assay based upon ICP-MS detection, we could monitor quantitatively cell adhesion induced by cell differentiation of THP-1 cells in response to phorbol ester stimulation (PMA) in single cell type or mixed cultures.
In this article we demonstrate that hybrid nanogels can be prepared by encapsulation of reactive ... more In this article we demonstrate that hybrid nanogels can be prepared by encapsulation of reactive nanoparticles (NPs) directly during nanogel synthesis. Nanogels investigated in present study consist of poly(N-vinylcaprolactam-co-(2-acetoacetoxyethyl) methacrylate) copolymer. ...
Phosphorus and sulfur are detected as phosphorus oxide and sulfur oxide ions (PO 1 and SO 1 ), pr... more Phosphorus and sulfur are detected as phosphorus oxide and sulfur oxide ions (PO 1 and SO 1 ), produced by oxidation reactions with O 2 performed in the reaction cell of the inductively coupled plasma dynamic reaction cell mass spectrometer (ICP-DRC-MS), at sub-ng mL 21 detection limits. This allows pM mL 21 detection of phospho-proteins, with S used as an internal standard (see ref. 9). The method was applied to digests (in HCl) of in-vitro tyrosine kinase assays, both as an evaluation of kinase autophosphorylation and phosphorylation of substrate. Detecting the phosphorus/sulfur ratio (via measured PO 1 /SO 1 ) in cell cultures is shown to provide a distinguishable difference between malignant cell lines and primary cultures. The PO 1 /SO 1 ratio for human colorectal adenocarcinoma (CRC) tissue samples compared with matched normal (N) tissue samples from the same patients is shown to be higher, at (PO 1 /SO 1 ) CRC /(PO 1 /SO 1 ) N~1 .75 ¡ 0.18 (n~4). Samples used in this analysis were of needle biopsy amounts (0.2-0.5 mg), with a greater than 70% tumor burden in CRC. The phospho-protein phosvitin is detected directly from dried one-dimensional polyacrylamide gels using laser ablation ICP-MS by detecting phosphorus at sub-nM amounts. The phosphorus detection limit for direct ablation, assessed from ablating gel doped with P and blank gel, is 0.6 mg g 21 in gel. Direct detection of sulfur from the gels is obscured by the high sulfur background for the blank gels, which is attributed to the sulfurcontaining catalysts used in polymerization and to sodium dodecyl sulfate (SDS) used for protein denaturing. 9 6 J . A n a l . A t . S p e c t r o m . , 2 0 0 4 , 1 9 , 9 6 -1 0 0 T h i s j o u r n a l i s ß T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
0267-9477(2010)25:3;1-F HOT PAPER Abdelrahman et al. Metal-containing polystyrene beads as standa... more 0267-9477(2010)25:3;1-F HOT PAPER Abdelrahman et al. Metal-containing polystyrene beads as standards for mass cytometry HOT PAPER Chan and Hieftje Algorithm to determine matrix-effect crossover points for overcoming interferences in inductively coupled plasma-atomic emission spectrometry
Advances in the development of highly multiplexed bio-analytical assays with inductively coupled ... more Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping.
150 kDa caldesmon was shown to be characteristic of vascular smooth muscle cells in normal tissue... more 150 kDa caldesmon was shown to be characteristic of vascular smooth muscle cells in normal tissue rather than in subculture. Subcultured smooth muscle cells from human aorta contained only the 70 kDa immunoreactive form of caldesmon. During the course of primary culture the amount of 150 kDa caldesmon as well as metavinculin decreased significantly whilst 70 kDa caldesmon became the predominant form, and by the onset of cell division the 150 kDa form was practically substituted by 70 kDa caldesmon. The data show that the predominance of 150 kDa caldesmon is characteristic of contractile smooth muscle cells, while in proliferating cells 70 kDa caldesmon is expressed.
A monoclonal antibody has been generated that interacts with the surface of cultured human aorta ... more A monoclonal antibody has been generated that interacts with the surface of cultured human aorta smooth muscle cells and does not bind to the endothelial cells from aorta and umbilical vein. An antigen recognized by the antibody has a molecular mass of 330 kDa as determined by electrophoresis of immunoprecipitate in SDS-polyacrylamide gel. The same antigen appeared to be present on the fibroblast surface while neither immunofluorescence, flow cytofluorimetry nor innnunoprecipitation reveal it on the endothelial cell surface or in the Triton X-100 extract. Smooth muscle cell Endothelial cell Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/85/$3.30 0 1985 Federation of European Biochemical, Societies
We have developed a monoclonal antibody that specifically interacts with a surface antigen of hum... more We have developed a monoclonal antibody that specifically interacts with a surface antigen of human fibroblasts and smooth muscle cells. The antibody (antibody IIGlO) recognizes a polypeptide of molecular mass 330,000, revealed by immunoblotting in fibroblast and smooth muscle cell extract, but not in vascular endothelial cells, peritoneal macrophages, peripheral blood lymphocytes nor hepatocytes. In tissue sections the antibody stained smooth muscle cells of myometrium, aorta and smaller blood vessels, and fibroblasts of connective tissue. Specificity of the antibody was further confirmed by double staining of aorta sections. Antibody IIGlO was used to identify smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta.
Molecular chaperones and cytosolic stress proteins are actively involved in the stabilization, im... more Molecular chaperones and cytosolic stress proteins are actively involved in the stabilization, import and refolding of precursor proteins into mitochondria. The purpose of the present study was to evaluate the relationship between mitochondrial content under steady-state conditions, and during the induction of organelle biogenesis, with the expression of stress proteins and mitochondrial chaperonins. A comparison of steady-state levels of mitochondrial enzyme activity [cytochrome c oxidase (CYTOX)] with chaperonin levels [the heat-shock protein HSP60, the glucose-regulated protein GRP75 (mtHSP70)] in striated muscles possessing a wide range of oxidative capacities revealed a proportional expression between the two. This relationship was disrupted by chronic contractile activity brought about by 10 days of 10 Hz stimulation of the tibialis anterior (TA) muscle, which induced 2.4-fold increases in CYTOX activity, but 3.2- and 9.3-fold increases in HSP60 and GRP75 respectively. The ind...
The control of enzyme activity by reversible phosphorylation and the role of protein kinases in s... more The control of enzyme activity by reversible phosphorylation and the role of protein kinases in signal transduction illustrate well the pivotal role of protein phosphorylation in cell regulation. There are numerous examples of the role of protein kinases and ...
The purpose of this phospho-proteomics study was to demonstrate the broad analysis of cellular pr... more The purpose of this phospho-proteomics study was to demonstrate the broad analysis of cellular protein phosphorylation in cells and tissue as a means to monitor changes in cellular states. As a cancer model, human tumor-derived A431 cells known to express the epidermal growth factor receptor (EGFR) were grown as cell cultures or xenograft tumors in mice. The cells and tumor-bearing animals were subjected to treatments including the EGFR-directed protein kinase inhibitor PK166 and/or EGF stimulation. Whole cell/tissue protein extracts were converted to peptides by using trypsin, and phosphorylated peptides were purified by an affinity capture method. Peptides and phosphorylation sites were characterized and quantified by using a combination of tandem mass spectroscopy (MS) and Fourier transform MS instrumentation (FTMS). By analyzing roughly 10 6 cell equivalents, 780 unique phosphopeptides from approx 450 different proteins were characterized. Only a small number of these phosphorylation sites have been described previously in literature. Although a targeted analysis of the EGFR pathway was not a specific aim of this study, 22 proteins known to be associated with EGFR signaling were identified. Fifty phosphopeptides were found changed in abundance as a function of growth factor or drug treatment including novel sites of phosphorylation on the EGFR itself. These findings demonstrate the feasibility of using phospho-proteomics to determine drug and disease mechanisms, and as a measure of drug target modulation in tissue.
We have developed a monoclonal antibody that specifically interacts with a surface antigen of hum... more We have developed a monoclonal antibody that specifically interacts with a surface antigen of human fibroblasts and smooth muscle cells. The antibody (antibody IIG10) recognizes a polypeptide of molecular mass 330,000, revealed by immunoblotting in fibroblast and smooth muscle cell extract, but not in vascular endothelial cells, peritoneal macrophages, peripheral blood lymphocytes nor hepatocytes. In tissue sections the antibody stained smooth muscle cells of myometrium, aorta and smaller blood vessels, and fibroblasts of connective tissue. Specificity of the antibody was further confirmed by double staining of aorta sections. Antibody IIG10 was used to identify smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta.
Mapping protein-protein interactions is an invaluable tool for understanding protein function. He... more Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.
Dystrophin, vinculin, and aciculin in skeletal muscle subject to chronic use and disuse REZVANI, ... more Dystrophin, vinculin, and aciculin in skeletal muscle subject to chronic use and disuse REZVANI, M OJGAN; ORNATSKY, OLGA I.; CONNOR, M ICHAEL K.; EISENBERG, HERBERT A.; HOOD, DAVID A.
We describe the synthesis and characterization of element-encoded polystyrene nanoparticles with ... more We describe the synthesis and characterization of element-encoded polystyrene nanoparticles with diameters on the order of 100 nm and a narrow size distribution. Individual particles contain ca. 10 3 chelated lanthanide ions, of either a single element or a mixture of elements. These particles were effectively internalized by nonspecific endocytosis into three cell lines associated with human leukemia. Using an assay based upon ICP-MS detection, we could monitor quantitatively cell adhesion induced by cell differentiation of THP-1 cells in response to phorbol ester stimulation (PMA) in single cell type or mixed cultures.
In this article we demonstrate that hybrid nanogels can be prepared by encapsulation of reactive ... more In this article we demonstrate that hybrid nanogels can be prepared by encapsulation of reactive nanoparticles (NPs) directly during nanogel synthesis. Nanogels investigated in present study consist of poly(N-vinylcaprolactam-co-(2-acetoacetoxyethyl) methacrylate) copolymer. ...
Phosphorus and sulfur are detected as phosphorus oxide and sulfur oxide ions (PO 1 and SO 1 ), pr... more Phosphorus and sulfur are detected as phosphorus oxide and sulfur oxide ions (PO 1 and SO 1 ), produced by oxidation reactions with O 2 performed in the reaction cell of the inductively coupled plasma dynamic reaction cell mass spectrometer (ICP-DRC-MS), at sub-ng mL 21 detection limits. This allows pM mL 21 detection of phospho-proteins, with S used as an internal standard (see ref. 9). The method was applied to digests (in HCl) of in-vitro tyrosine kinase assays, both as an evaluation of kinase autophosphorylation and phosphorylation of substrate. Detecting the phosphorus/sulfur ratio (via measured PO 1 /SO 1 ) in cell cultures is shown to provide a distinguishable difference between malignant cell lines and primary cultures. The PO 1 /SO 1 ratio for human colorectal adenocarcinoma (CRC) tissue samples compared with matched normal (N) tissue samples from the same patients is shown to be higher, at (PO 1 /SO 1 ) CRC /(PO 1 /SO 1 ) N~1 .75 ¡ 0.18 (n~4). Samples used in this analysis were of needle biopsy amounts (0.2-0.5 mg), with a greater than 70% tumor burden in CRC. The phospho-protein phosvitin is detected directly from dried one-dimensional polyacrylamide gels using laser ablation ICP-MS by detecting phosphorus at sub-nM amounts. The phosphorus detection limit for direct ablation, assessed from ablating gel doped with P and blank gel, is 0.6 mg g 21 in gel. Direct detection of sulfur from the gels is obscured by the high sulfur background for the blank gels, which is attributed to the sulfurcontaining catalysts used in polymerization and to sodium dodecyl sulfate (SDS) used for protein denaturing. 9 6 J . A n a l . A t . S p e c t r o m . , 2 0 0 4 , 1 9 , 9 6 -1 0 0 T h i s j o u r n a l i s ß T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
0267-9477(2010)25:3;1-F HOT PAPER Abdelrahman et al. Metal-containing polystyrene beads as standa... more 0267-9477(2010)25:3;1-F HOT PAPER Abdelrahman et al. Metal-containing polystyrene beads as standards for mass cytometry HOT PAPER Chan and Hieftje Algorithm to determine matrix-effect crossover points for overcoming interferences in inductively coupled plasma-atomic emission spectrometry
Advances in the development of highly multiplexed bio-analytical assays with inductively coupled ... more Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping.
150 kDa caldesmon was shown to be characteristic of vascular smooth muscle cells in normal tissue... more 150 kDa caldesmon was shown to be characteristic of vascular smooth muscle cells in normal tissue rather than in subculture. Subcultured smooth muscle cells from human aorta contained only the 70 kDa immunoreactive form of caldesmon. During the course of primary culture the amount of 150 kDa caldesmon as well as metavinculin decreased significantly whilst 70 kDa caldesmon became the predominant form, and by the onset of cell division the 150 kDa form was practically substituted by 70 kDa caldesmon. The data show that the predominance of 150 kDa caldesmon is characteristic of contractile smooth muscle cells, while in proliferating cells 70 kDa caldesmon is expressed.
A monoclonal antibody has been generated that interacts with the surface of cultured human aorta ... more A monoclonal antibody has been generated that interacts with the surface of cultured human aorta smooth muscle cells and does not bind to the endothelial cells from aorta and umbilical vein. An antigen recognized by the antibody has a molecular mass of 330 kDa as determined by electrophoresis of immunoprecipitate in SDS-polyacrylamide gel. The same antigen appeared to be present on the fibroblast surface while neither immunofluorescence, flow cytofluorimetry nor innnunoprecipitation reveal it on the endothelial cell surface or in the Triton X-100 extract. Smooth muscle cell Endothelial cell Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/85/$3.30 0 1985 Federation of European Biochemical, Societies
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Papers by Olga Ornatsky