- Email: [email protected]
Proteomics and Genomics
PROTEOMICS AND GENOMICS
11 - Proteomics and Genomics 321 Protective Effects of S-Adenosylmethionine in Alcohol Induced Fatty Liver Disease: Identification of Molecular Targets in the Mitochondrial Proteome Kelly K Andringa1, Sudheer K. Mantena1, and Shannon M. Bailey1 1 University of Alabama at Birmingham S-adenosylmethionine (SAM) has shown benefits in treating alcoholic and nonalcoholic liver disease. Previously, we reported that SAM co-administration prevents chronic alcohol induced steatosis, mitochondrial dysfunction, and oxidative stress; however, the molecular mechanisms responsible for SAM hepatoprotection remain unknown. Herein, we analyze alterations in the liver mitochondrial proteome caused by chronic alcohol consumption and SAM treatment. Male rats were pair-fed control or alcohol-containing liquid diets with or without SAM (0.8 mg/mL diet) for 5 wks. Liver tissue was prepared for histopathology and mitochondrial proteome analysis. Two dimension isoelectric focusing/SDS-PAGE (2D IEF) proteomics along with blue native electrophoresis (BN-PAGE) were used to determine changes in matrix and oxidative phosphorylation (OxPhos) proteins. SAM coadministration decreased micro- and macrovesicular fat accumulation in liver of alcohol-fed rats whereas no pathology was seen in control or control + SAM groups. in 2D IEF studies, 76 mitochondrial proteins were matched from gels of the four treatment groups (control; alcohol; control + SAM; alcohol + SAM) with 30 proteins showing significant changes in response to alcohol and/or SAM. the proteins most affected by chronic alcohol consumption are members of key functional groups including chaperones, fatty acid oxidation proteins, sulfur amino acid metabolism proteins, and oxidoreductase enzymes. Importantly, SAM prevented many of the alcohol-induced changes in protein abundance. Using BN-PAGE, we observed significant decreases in 17 OxPhos proteins in response to alcohol, with SAM largely preventing these changes. Complexes I and IV showed the most change in response to alcohol and/or SAM treatment. These data indicate that the hepatoprotective effects of SAM in treating alcohol-induced liver disease may be mediated through modifications to the mitochondrial proteome. Investigating the effects of SAM on metabolic networks in the mitochondrial proteome will help identify the mechanisms involved in SAM hepatoprotection and thereby facilitate the development of new therapeutics for alcoholic fatty liver disease treatment.
322 Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 From Saccharomyces cerevisiae Karen Fulan Discola1, Marcos Antonio Oliveira2, Gisele Monteiro1, Beatriz Gomes Guimarães3, and Luis Eduardo Soares Netto1 1 2 Universidade de São Paulo, São Paulo, Brazil, Universidade 3 Estadual Paulista, São Vicente, Brazil, Synchrotron SOLEIL France Glutaredoxins (Grxs) are small heat stable thiol-dependent oxidoreductases with at least one cysteine at their active sites. Although Grxs are implicated in many cellular processes, including sulfur metabolism, protein folding and protection against reactive oxygen species, their targets are known. in Saccharomyces cerevisiae, seven Grxs isoforms were identified (Grx 1-7). Grx1-2 are dithiol glutaredoxins which contains the conserved CPYC motif in their active sites, whereas Grx3-7 are monothiolic isoforms. Both dithiol glutaredoxins are cytosolic, however Grx2 is also located at the mitochondria. in spite of the
fact that Grx1 and Grx2 share 85% of amino acid sequence similarity, Grx2 was fifteen times more active as oxidoreductase than Grx1. in an attempt to better understand the mechanisms and differences between yeast dithiol Grxs activities, we elucidated the crystallographic structures of yeast Grx2 in the oxidized state and of the Grx2-C30S mutant with glutathionyl mixed disulfide. Comparisons among these structures and those of yeast Grx1 (Hakansson & Winther, 2007) provided insights into the remarkable functional divergence between these enzymes. 23 52 We hypothesize that the substitutions of Ser and Gln in yGrx1 23 52 by Ala and Glu in yGrx2 can modify the capability of the active site C-terminal cysteine to attack the mixed disulfide between the N-terminal active site cysteine and the glutathione molecule. 89 89 Furthermore, the presence of Asp in yGrx1 instead of Ser in yGrx2 could be related to differences in the glutathione affinity. These hypotheses are being tested by site-specific mutagenesis studies.
323 Vitamin C Promotes Dermal Fibroblast Proliferation, Migration and Replication-Associated Base Excision Repair of Oxidised DNA Bases: a New Protective Role for Vitamin C in the Skin? Tiago L Duarte1, Marcus S Cooke2, and George DD Jones2 1 Instituto de Biologia Molecular e Celular, Universidade do Porto, 2 Portugal, University of Leicester, UK The skin is a protective barrier against external insults. One such insult is UV radiation, which is known to react with cellular photosensitisers and lead to the formation of reactive oxygen species. Vitamin C is not only an important free radical scavenger, but also an essential co-factor for collagen synthesis by dermal fibroblasts, and therefore plays an important role in skin homeostasis. Here, we investigated the ability of vitamin C to modulate cellular repair of oxidatively modified DNA bases. Contact-inhibited populations of human dermal fibroblasts were exposed to a singlet oxygen generating system (Ro 19-8022 photosensitiser plus light) and the repair of 7,8-dihydro-8-oxoguanine was monitored using the alkaline comet assay in conjunction with hOGG1 endonuclease. Notably, cells that were pre-incubated with a stable vitamin C derivative, ascorbic acid 2phosphate (AA2P), showed faster repair kinetics. to investigate if long-term exposure to AA2P would result in the up-regulation of specific DNA repair pathways, we identified AA2P-responsive genes using oligonucleotide microarray and quantitative RT-PCR. We observed an increase in the expression of genes associated with DNA replication and repair, and with G2/M phase of the cell cycle, as well as a reduction in the expression of genes involved in fibroblast differentiation and glucose metabolism. Consistent with the gene expression changes, we observed that AA2P treatment increased cell cycle re-entry of quiescent fibroblasts in response to serum stimulation and cell motility (wound healing capacity). We propose that, in addition to its important antioxidant role, vitamin C may protect the skin and accelerate wound healing by promoting fibroblast proliferation, migration and replicationassociated base excision repair of potentially mutagenic DNA lesions.
324 Gene and Protein Responses to Extracellular Redox Potential Young-Mi Go1, Siobhan Moriarty-Craige2, and Dean P. Jones1 1 2 Emory University, University of Massachusetts Extracellular cysteine (Cys) and cystine (CySS) redox potential
SFRBM2008
S121
(Eh) controls cell death, proliferation, and inflammation, suggesting that these functions are regulated by redox sensitive molecules. However, changes in gene and protein expressions due to extracellular redox potential are largely unknown. in the present study, we identify cellular changes of human THP-1 monocytes, which are continuously exposed to either reduced or oxidized extracellular Cys/CySS redox potential. a global approach was undertaken to examine gene and protein changes after 36 h of reduced versus oxidized extracellular redox conditions. Microarray analysis identified 70 genes which were more than 2-fold different between the oxidized and reduced Eh. of these, 44 were upregulated by the oxidized Eh. Ingenuity pathway analysis (IPA)-defined the main categories were cell death, cell growth and proliferation, and stress/detoxification (xenobiotic metabolism). 4 genes [ATP binding cassett, aldo-keto reductase family 1, NAD(P)H dehydrogenase, quinone 1 (NQO1), prostaglandin-endoperoxide synthase 2] were quantitatively analyzed by real-time PCR and verified microarray data. We also examined changes in protein abundance by isotope coded affinity tag (ICAT)-based nanoLC-MS/MS proteomic analysis. the ICAT analysis identified 115 proteins that were more than 20% increased in abundance in the oxidized condition. IPA-defined top networks were protein synthesis, cell growth and proliferation, and cellular assembly and organization. the top canonical pathway responding to oxidized Eh was defined as the oxidative stress response mediated by Nrf2. This was consistent with the results by gene array analysis, which also showed upregulation of Nrf2dependent genes, ferritin heavy and light peptide 1, v-maf, and NQO1. Together, the findings suggest that cell survival and proliferative functions can be mediated through gene expression and protein abundance signalled by extracellular redox potential.
325 DIGE Analysis of the S-Nitroso Proteome in Airway Epithelial Cells Nicholas Kettenhofen1, and Neil Hogg1 1 Medical College of Wisconsin Post-translational modifications of cysteine residues have been proposed to be critical events in cellular redox signaling pathways given their potential to reversibly alter protein function. Protein Snitrosation (PrSNO) in particular has been suggested as a mechanism by which nitric oxide (NO) can alter cellular function through thiol modifications. However, proteomic studies designed to identify specific protein targets in biological systems have proven difficult. the position of the airway epithelium as a direct environmental interface makes it an interesting system in which to analyze nitrosative and oxidative protein modifications. A combination of CyDye switch labeling and differential in-gel electrophoresis (DIGE) was used to determine the S-nitroso proteome in cultured human bronchial epithelial cells following exposure to nitrosative stress. Cells were treated with varying concentrations of the NO-donating agent, spermine NONOate, or the transportable low-molecular weight S-nitrosothiol, Snitrosocysteine (CysNO). Total intracellular PrSNO levels were then quantified by tri-iodide chemiluminescence in order to provide a relative biological context for the proteomic analyses. CyDye switch labeling selectively replaced the protein cysteinyl nitroso group with one of two maleimide-based fluorescent dyes. Distinct fluorophores (Cy3 and Cy5) facilitated DIGE comparison of identically-processed control and treated samples, labeled with different dyes, and co-separated within a single 2D gel. Fluorescence imaging and software analysis uncovered PrSNO candidate spots that presented differential labeling and proteins were subsequently identified using MALDI-TOF peptide mass fingerprinting. Similar proteins were found to be modified when equivalent cellular PrSNO levels were generated by both NO and CysNO treatments. However, approximately 400 times more NO
S122
(mole:mole) was required to match the levels of PrSNO formed by CysNO. Spot pattern analysis performed as a function of decreasing total intracellular PrSNO level revealed that only a subset of proteins remains modified at low cellular PrSNO levels, suggesting a hierarchy of target sensitivity.
326 Profiling of the Human Plasma Oxidized Proteome Ashraf G. Madian1, and Fred E. Regnier1 1 Purdue University Oxidative stress plays an important role in the changes that occur during aging and a large number of diseases. Oxidative damage inside cells leaves a universal fingerprint in the form of protein carbonylation, often leading to the loss of the protein function. Proteins can be carbonylated by glycation of lysine and oxidation of arginine, threonine, lysine or proline. Aldehydes generated from lipid peroxidation can react with proteins to form carbonyl groups as well. In this study, we report the first proteomic based identification and characterization of oxidized proteins in human plasma. Biotin hydrazide was added to four human plasma samples (age 20-40). and the resulting Schiff’s bases were reduced by sodium caynoborohydride. Proteins thus biotinylated were affinity enriched by immobilized avidin beads. the selected proteins were digested with trypsin and the peptide fragments were separated by the C18 reversed phase chromatography, identified and characterized by both Electrospray (ESI) and Matrix Assisted Laser Desorption Ionization (MALDI) based mass spectrometers. Nearly 3% of the total protein content in plasma was found to be carbonylated. Thirty proteins were identified in all the four subjects used. Many of the oxidized proteins identified had not previously been shown to be oxidized. Oxidative modifications (e.g kynurenin, pyroglutamic and aminoadipic semialdehyde) were identified in fourteen proteins. Kidney, Lung , liver and soft tissues contributed most of the oxidized proteins followed by cerebral cortex and heart muscle.
327 Fluorescent Tagging Strategy for Oxidized Tyrosines in Aging Cardiac Tissue Maria B. Thorson1, Sung Jung Hong1, Victor S. Sharov1, Elena S. Dremina1, Jacque Killmer1, Justin P. Pennington1, Xiaobao Li1, John F. Stobaugh1, and Christian Schoeneich1 1 The University of Kansas Purpose: for the labeling, enrichment, identification, and relative quantification of the protein oxidation products 3-hydroxytyrosine (3,4-dihydroxyphenylalanine, DOPA) and 3-nitrotyrosine (3NY), a method using benzylamine-dependent chemistry is being developed. Successful labeling of model compounds has been observed, and the present study applies the method to cardiac tissue samples from both old and young rats in order to study age-dependent differences in protein oxidation in vivo. Methods: Soluble proteins from old and young rat cardiac tissue are reacted with excess benzylamine and potassium ferricyanide to fluorescently label DOPA residues by forming 2phenylbenzoxazole-related compounds. to label 3NY residues, the proteins are first reduced to 3-aminotyrosine (3AY), which can undergo a similar reaction with benzylamine and oxidant to give the same products. Nano-LC-MS/MS (FT-ICR or ion trap) is used to identify specific sites of in-vivo oxidative modification. Model peptides hydroxylated by tyrosinase or synthesized with the 3NY residue are labeled with the same chemistry and used to optimize reaction conditions and study the effect of amino acid sequence
SFRBM2008
on labeling. Results: Proteins from cardiac tissue have been fluorescently labeled and separated. Some putative identifications have been made by mass spectrometry. Reagent concentration dependences, pH dependence, and kinetics have been studied in model systems. Conclusions: This method aids identification of the in-vivo protein oxidation products DOPA and 3NY in tissue samples and can also be adapted for affinity enrichment and relative quantification of these low-abundance species.
of human health. We have found that human red blood cells have about 180,000 copies of active, tetrameric catalase per cell; a typical HL-60 cell has about 500,000 copies of catalase; this number varies with cell density, the greater the cell density at harvest, the greater the number of copies of catalase. Our long term goal is to apportion the removal of hydrogen peroxide from cells among the various enzymatic pathways so as to dissect the role of each pathway in the basic biology of the cell. (Supported by NIH 1R01GM073929 and NIH 5T32CA078586)
328 Discovery and Targeted Assay Development for the Biomarkers of Cisplatin Resistance in Human Ovarian Cancer Using Mass Spectrometry Mu Wang1,2 1 2 Monarch LifeSciences, Indiana University School of Medicine Platinum-based chemotherapy, such as cisplatin, is the primary treatment for ovarian cancer. However, drug resistance has become a major impediment to the successful treatment of ovarian cancer. to date, the molecular mechanisms of resistance to platinum-based chemotherapy remain unclear. in this study, we applied a LC/MS-based, label-free, protein quantification method to examine the global protein expression profiles of two pairs of ovarian cancer cell lines, A2780/A2780-CP (cisplatinsensitive/cisplatin-resistant) and 2008/2008-C13*5.25 (cisplatinsensitive/cisplatin-resistant). We identified and quantified over 2000 proteins from these cell lines and 95 proteins showed significant expression changes between sensitive and resistant groups with a false-discovery-rate (FDR) of less than 5%. Bioinformatics analysis suggested several potential pathways that may be involved in cisplatin resistance. Among these potential pathways, a redox regulated pathway involving superoxide dismutase 1 (SOD1) was targeted in order to further explore its involvement in drug resistance. SOD 1 represents a potential biomarker for early evaluation of drug resistance. Inhibition of SOD1 expression and/or activity enabled re-sensitization of the cisplatin resistant ovarian cancer cells. This study provides not only a new proteomic platform for largescale quantitative protein analysis, but also important information for potential biomarkers of cisplatin resistance in ovarian cancer. Furthermore, these results may be clinically relevant for diagnostics, prognostics, and therapeutic improvement for ovarian cancer treatment.
329 Absolute Quantitation of Catalase in Mammalian Cells: Changes Seen With Cell Density Jordan R Witmer1, Brett a Wagner1, and Garry R Buettner1 1 The University of Iowa The expression of antioxidant enzymes varies among cell types; even within a cell type the amount of some of these enzymes can vary considerably as a function of the biological state of the cell. We have developed a method to determine on an absolute basis the number of active catalase enzymes in a cell. Knowing the number of catalase enzymes in various cells is our first step in the absolute quantitation of the many redox active species and antioxidant enzymes in cells. This information will open the door to in silico approaches that can be used to complement laboratory data, bringing greater understanding to basic biology and issues
SFRBM2008
S123
11 - Proteomics and Genomics 321 Protective Effects of S-Adenosylmethionine in Alcohol Induced Fatty Liver Disease: Identification of Molecular Targets in the Mitochondrial Proteome Kelly K Andringa1, Sudheer K. Mantena1, and Shannon M. Bailey1 1 University of Alabama at Birmingham S-adenosylmethionine (SAM) has shown benefits in treating alcoholic and nonalcoholic liver disease. Previously, we reported that SAM co-administration prevents chronic alcohol induced steatosis, mitochondrial dysfunction, and oxidative stress; however, the molecular mechanisms responsible for SAM hepatoprotection remain unknown. Herein, we analyze alterations in the liver mitochondrial proteome caused by chronic alcohol consumption and SAM treatment. Male rats were pair-fed control or alcohol-containing liquid diets with or without SAM (0.8 mg/mL diet) for 5 wks. Liver tissue was prepared for histopathology and mitochondrial proteome analysis. Two dimension isoelectric focusing/SDS-PAGE (2D IEF) proteomics along with blue native electrophoresis (BN-PAGE) were used to determine changes in matrix and oxidative phosphorylation (OxPhos) proteins. SAM coadministration decreased micro- and macrovesicular fat accumulation in liver of alcohol-fed rats whereas no pathology was seen in control or control + SAM groups. in 2D IEF studies, 76 mitochondrial proteins were matched from gels of the four treatment groups (control; alcohol; control + SAM; alcohol + SAM) with 30 proteins showing significant changes in response to alcohol and/or SAM. the proteins most affected by chronic alcohol consumption are members of key functional groups including chaperones, fatty acid oxidation proteins, sulfur amino acid metabolism proteins, and oxidoreductase enzymes. Importantly, SAM prevented many of the alcohol-induced changes in protein abundance. Using BN-PAGE, we observed significant decreases in 17 OxPhos proteins in response to alcohol, with SAM largely preventing these changes. Complexes I and IV showed the most change in response to alcohol and/or SAM treatment. These data indicate that the hepatoprotective effects of SAM in treating alcohol-induced liver disease may be mediated through modifications to the mitochondrial proteome. Investigating the effects of SAM on metabolic networks in the mitochondrial proteome will help identify the mechanisms involved in SAM hepatoprotection and thereby facilitate the development of new therapeutics for alcoholic fatty liver disease treatment.
322 Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 From Saccharomyces cerevisiae Karen Fulan Discola1, Marcos Antonio Oliveira2, Gisele Monteiro1, Beatriz Gomes Guimarães3, and Luis Eduardo Soares Netto1 1 2 Universidade de São Paulo, São Paulo, Brazil, Universidade 3 Estadual Paulista, São Vicente, Brazil, Synchrotron SOLEIL France Glutaredoxins (Grxs) are small heat stable thiol-dependent oxidoreductases with at least one cysteine at their active sites. Although Grxs are implicated in many cellular processes, including sulfur metabolism, protein folding and protection against reactive oxygen species, their targets are known. in Saccharomyces cerevisiae, seven Grxs isoforms were identified (Grx 1-7). Grx1-2 are dithiol glutaredoxins which contains the conserved CPYC motif in their active sites, whereas Grx3-7 are monothiolic isoforms. Both dithiol glutaredoxins are cytosolic, however Grx2 is also located at the mitochondria. in spite of the
fact that Grx1 and Grx2 share 85% of amino acid sequence similarity, Grx2 was fifteen times more active as oxidoreductase than Grx1. in an attempt to better understand the mechanisms and differences between yeast dithiol Grxs activities, we elucidated the crystallographic structures of yeast Grx2 in the oxidized state and of the Grx2-C30S mutant with glutathionyl mixed disulfide. Comparisons among these structures and those of yeast Grx1 (Hakansson & Winther, 2007) provided insights into the remarkable functional divergence between these enzymes. 23 52 We hypothesize that the substitutions of Ser and Gln in yGrx1 23 52 by Ala and Glu in yGrx2 can modify the capability of the active site C-terminal cysteine to attack the mixed disulfide between the N-terminal active site cysteine and the glutathione molecule. 89 89 Furthermore, the presence of Asp in yGrx1 instead of Ser in yGrx2 could be related to differences in the glutathione affinity. These hypotheses are being tested by site-specific mutagenesis studies.
323 Vitamin C Promotes Dermal Fibroblast Proliferation, Migration and Replication-Associated Base Excision Repair of Oxidised DNA Bases: a New Protective Role for Vitamin C in the Skin? Tiago L Duarte1, Marcus S Cooke2, and George DD Jones2 1 Instituto de Biologia Molecular e Celular, Universidade do Porto, 2 Portugal, University of Leicester, UK The skin is a protective barrier against external insults. One such insult is UV radiation, which is known to react with cellular photosensitisers and lead to the formation of reactive oxygen species. Vitamin C is not only an important free radical scavenger, but also an essential co-factor for collagen synthesis by dermal fibroblasts, and therefore plays an important role in skin homeostasis. Here, we investigated the ability of vitamin C to modulate cellular repair of oxidatively modified DNA bases. Contact-inhibited populations of human dermal fibroblasts were exposed to a singlet oxygen generating system (Ro 19-8022 photosensitiser plus light) and the repair of 7,8-dihydro-8-oxoguanine was monitored using the alkaline comet assay in conjunction with hOGG1 endonuclease. Notably, cells that were pre-incubated with a stable vitamin C derivative, ascorbic acid 2phosphate (AA2P), showed faster repair kinetics. to investigate if long-term exposure to AA2P would result in the up-regulation of specific DNA repair pathways, we identified AA2P-responsive genes using oligonucleotide microarray and quantitative RT-PCR. We observed an increase in the expression of genes associated with DNA replication and repair, and with G2/M phase of the cell cycle, as well as a reduction in the expression of genes involved in fibroblast differentiation and glucose metabolism. Consistent with the gene expression changes, we observed that AA2P treatment increased cell cycle re-entry of quiescent fibroblasts in response to serum stimulation and cell motility (wound healing capacity). We propose that, in addition to its important antioxidant role, vitamin C may protect the skin and accelerate wound healing by promoting fibroblast proliferation, migration and replicationassociated base excision repair of potentially mutagenic DNA lesions.
324 Gene and Protein Responses to Extracellular Redox Potential Young-Mi Go1, Siobhan Moriarty-Craige2, and Dean P. Jones1 1 2 Emory University, University of Massachusetts Extracellular cysteine (Cys) and cystine (CySS) redox potential
SFRBM2008
S121
(Eh) controls cell death, proliferation, and inflammation, suggesting that these functions are regulated by redox sensitive molecules. However, changes in gene and protein expressions due to extracellular redox potential are largely unknown. in the present study, we identify cellular changes of human THP-1 monocytes, which are continuously exposed to either reduced or oxidized extracellular Cys/CySS redox potential. a global approach was undertaken to examine gene and protein changes after 36 h of reduced versus oxidized extracellular redox conditions. Microarray analysis identified 70 genes which were more than 2-fold different between the oxidized and reduced Eh. of these, 44 were upregulated by the oxidized Eh. Ingenuity pathway analysis (IPA)-defined the main categories were cell death, cell growth and proliferation, and stress/detoxification (xenobiotic metabolism). 4 genes [ATP binding cassett, aldo-keto reductase family 1, NAD(P)H dehydrogenase, quinone 1 (NQO1), prostaglandin-endoperoxide synthase 2] were quantitatively analyzed by real-time PCR and verified microarray data. We also examined changes in protein abundance by isotope coded affinity tag (ICAT)-based nanoLC-MS/MS proteomic analysis. the ICAT analysis identified 115 proteins that were more than 20% increased in abundance in the oxidized condition. IPA-defined top networks were protein synthesis, cell growth and proliferation, and cellular assembly and organization. the top canonical pathway responding to oxidized Eh was defined as the oxidative stress response mediated by Nrf2. This was consistent with the results by gene array analysis, which also showed upregulation of Nrf2dependent genes, ferritin heavy and light peptide 1, v-maf, and NQO1. Together, the findings suggest that cell survival and proliferative functions can be mediated through gene expression and protein abundance signalled by extracellular redox potential.
325 DIGE Analysis of the S-Nitroso Proteome in Airway Epithelial Cells Nicholas Kettenhofen1, and Neil Hogg1 1 Medical College of Wisconsin Post-translational modifications of cysteine residues have been proposed to be critical events in cellular redox signaling pathways given their potential to reversibly alter protein function. Protein Snitrosation (PrSNO) in particular has been suggested as a mechanism by which nitric oxide (NO) can alter cellular function through thiol modifications. However, proteomic studies designed to identify specific protein targets in biological systems have proven difficult. the position of the airway epithelium as a direct environmental interface makes it an interesting system in which to analyze nitrosative and oxidative protein modifications. A combination of CyDye switch labeling and differential in-gel electrophoresis (DIGE) was used to determine the S-nitroso proteome in cultured human bronchial epithelial cells following exposure to nitrosative stress. Cells were treated with varying concentrations of the NO-donating agent, spermine NONOate, or the transportable low-molecular weight S-nitrosothiol, Snitrosocysteine (CysNO). Total intracellular PrSNO levels were then quantified by tri-iodide chemiluminescence in order to provide a relative biological context for the proteomic analyses. CyDye switch labeling selectively replaced the protein cysteinyl nitroso group with one of two maleimide-based fluorescent dyes. Distinct fluorophores (Cy3 and Cy5) facilitated DIGE comparison of identically-processed control and treated samples, labeled with different dyes, and co-separated within a single 2D gel. Fluorescence imaging and software analysis uncovered PrSNO candidate spots that presented differential labeling and proteins were subsequently identified using MALDI-TOF peptide mass fingerprinting. Similar proteins were found to be modified when equivalent cellular PrSNO levels were generated by both NO and CysNO treatments. However, approximately 400 times more NO
S122
(mole:mole) was required to match the levels of PrSNO formed by CysNO. Spot pattern analysis performed as a function of decreasing total intracellular PrSNO level revealed that only a subset of proteins remains modified at low cellular PrSNO levels, suggesting a hierarchy of target sensitivity.
326 Profiling of the Human Plasma Oxidized Proteome Ashraf G. Madian1, and Fred E. Regnier1 1 Purdue University Oxidative stress plays an important role in the changes that occur during aging and a large number of diseases. Oxidative damage inside cells leaves a universal fingerprint in the form of protein carbonylation, often leading to the loss of the protein function. Proteins can be carbonylated by glycation of lysine and oxidation of arginine, threonine, lysine or proline. Aldehydes generated from lipid peroxidation can react with proteins to form carbonyl groups as well. In this study, we report the first proteomic based identification and characterization of oxidized proteins in human plasma. Biotin hydrazide was added to four human plasma samples (age 20-40). and the resulting Schiff’s bases were reduced by sodium caynoborohydride. Proteins thus biotinylated were affinity enriched by immobilized avidin beads. the selected proteins were digested with trypsin and the peptide fragments were separated by the C18 reversed phase chromatography, identified and characterized by both Electrospray (ESI) and Matrix Assisted Laser Desorption Ionization (MALDI) based mass spectrometers. Nearly 3% of the total protein content in plasma was found to be carbonylated. Thirty proteins were identified in all the four subjects used. Many of the oxidized proteins identified had not previously been shown to be oxidized. Oxidative modifications (e.g kynurenin, pyroglutamic and aminoadipic semialdehyde) were identified in fourteen proteins. Kidney, Lung , liver and soft tissues contributed most of the oxidized proteins followed by cerebral cortex and heart muscle.
327 Fluorescent Tagging Strategy for Oxidized Tyrosines in Aging Cardiac Tissue Maria B. Thorson1, Sung Jung Hong1, Victor S. Sharov1, Elena S. Dremina1, Jacque Killmer1, Justin P. Pennington1, Xiaobao Li1, John F. Stobaugh1, and Christian Schoeneich1 1 The University of Kansas Purpose: for the labeling, enrichment, identification, and relative quantification of the protein oxidation products 3-hydroxytyrosine (3,4-dihydroxyphenylalanine, DOPA) and 3-nitrotyrosine (3NY), a method using benzylamine-dependent chemistry is being developed. Successful labeling of model compounds has been observed, and the present study applies the method to cardiac tissue samples from both old and young rats in order to study age-dependent differences in protein oxidation in vivo. Methods: Soluble proteins from old and young rat cardiac tissue are reacted with excess benzylamine and potassium ferricyanide to fluorescently label DOPA residues by forming 2phenylbenzoxazole-related compounds. to label 3NY residues, the proteins are first reduced to 3-aminotyrosine (3AY), which can undergo a similar reaction with benzylamine and oxidant to give the same products. Nano-LC-MS/MS (FT-ICR or ion trap) is used to identify specific sites of in-vivo oxidative modification. Model peptides hydroxylated by tyrosinase or synthesized with the 3NY residue are labeled with the same chemistry and used to optimize reaction conditions and study the effect of amino acid sequence
SFRBM2008
on labeling. Results: Proteins from cardiac tissue have been fluorescently labeled and separated. Some putative identifications have been made by mass spectrometry. Reagent concentration dependences, pH dependence, and kinetics have been studied in model systems. Conclusions: This method aids identification of the in-vivo protein oxidation products DOPA and 3NY in tissue samples and can also be adapted for affinity enrichment and relative quantification of these low-abundance species.
of human health. We have found that human red blood cells have about 180,000 copies of active, tetrameric catalase per cell; a typical HL-60 cell has about 500,000 copies of catalase; this number varies with cell density, the greater the cell density at harvest, the greater the number of copies of catalase. Our long term goal is to apportion the removal of hydrogen peroxide from cells among the various enzymatic pathways so as to dissect the role of each pathway in the basic biology of the cell. (Supported by NIH 1R01GM073929 and NIH 5T32CA078586)
328 Discovery and Targeted Assay Development for the Biomarkers of Cisplatin Resistance in Human Ovarian Cancer Using Mass Spectrometry Mu Wang1,2 1 2 Monarch LifeSciences, Indiana University School of Medicine Platinum-based chemotherapy, such as cisplatin, is the primary treatment for ovarian cancer. However, drug resistance has become a major impediment to the successful treatment of ovarian cancer. to date, the molecular mechanisms of resistance to platinum-based chemotherapy remain unclear. in this study, we applied a LC/MS-based, label-free, protein quantification method to examine the global protein expression profiles of two pairs of ovarian cancer cell lines, A2780/A2780-CP (cisplatinsensitive/cisplatin-resistant) and 2008/2008-C13*5.25 (cisplatinsensitive/cisplatin-resistant). We identified and quantified over 2000 proteins from these cell lines and 95 proteins showed significant expression changes between sensitive and resistant groups with a false-discovery-rate (FDR) of less than 5%. Bioinformatics analysis suggested several potential pathways that may be involved in cisplatin resistance. Among these potential pathways, a redox regulated pathway involving superoxide dismutase 1 (SOD1) was targeted in order to further explore its involvement in drug resistance. SOD 1 represents a potential biomarker for early evaluation of drug resistance. Inhibition of SOD1 expression and/or activity enabled re-sensitization of the cisplatin resistant ovarian cancer cells. This study provides not only a new proteomic platform for largescale quantitative protein analysis, but also important information for potential biomarkers of cisplatin resistance in ovarian cancer. Furthermore, these results may be clinically relevant for diagnostics, prognostics, and therapeutic improvement for ovarian cancer treatment.
329 Absolute Quantitation of Catalase in Mammalian Cells: Changes Seen With Cell Density Jordan R Witmer1, Brett a Wagner1, and Garry R Buettner1 1 The University of Iowa The expression of antioxidant enzymes varies among cell types; even within a cell type the amount of some of these enzymes can vary considerably as a function of the biological state of the cell. We have developed a method to determine on an absolute basis the number of active catalase enzymes in a cell. Knowing the number of catalase enzymes in various cells is our first step in the absolute quantitation of the many redox active species and antioxidant enzymes in cells. This information will open the door to in silico approaches that can be used to complement laboratory data, bringing greater understanding to basic biology and issues
SFRBM2008
S123