Publications (4)20.6 Total impact
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Article: A robust method for quantitative high-throughput analysis of proteomes by 18O labeling.
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ABSTRACT: MS-based quantitative proteomics plays an increasingly important role in biological and medical research and the development of these techniques remains one of the most important challenges in mass spectrometry. Numerous stable isotope labeling approaches have been proposed. However, and particularly in the case of (18)O-labeling, a standard protocol of general applicability is still lacking, and statistical issues associated to these methods remain to be investigated. In this work we present an improved high-throughput quantitative proteomics method based on whole proteome concentration by SDS-PAGE, optimized in-gel digestion, peptide (18)O-labeling, and separation by off-gel isoelectric focusing followed by liquid chromatography-LIT-MS. We demonstrate that the off-gel technique is fully compatible with (18)O peptide labeling in any pH range. A recently developed statistical model indicated that partial digestions and methionine oxidation do not alter protein quantification and that variances at the scan, peptide, and protein levels are stable and reproducible in a variety of proteomes of different origin. We have also analyzed the dynamic range of quantification and demonstrated the practical utility of the method by detecting expression changes in a model of activation of Jurkat T-cells. Our protocol provides a general approach to perform quantitative proteomics by (18)O-labeling in high-throughput studies, with the added value that it has a validated statistical model for the null hypothesis. To the best of our knowledge, this is the first report where a general protocol for stable isotope labeling is tested in practice using a collection of samples and analyzed at this degree of statistical detail.Molecular & Cellular Proteomics 01/2011; 10(1):M110.003335. · 7.40 Impact Factor -
Article: Cyclosporine A-induced nitration of tyrosine 34 MnSOD in endothelial cells: role of mitochondrial superoxide.
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ABSTRACT: Cyclosporine A (CsA) has represented a fundamental therapeutic weapon in immunosuppression for the past three decades. However, its clinical use is not devoid of side effects, among which hypertension and vascular injury represent a major drawback. Endothelial cells are able to generate reactive oxygen and nitrogen species upon exposure to CsA, including formation of peroxynitrite. This may result in endothelial cell toxicity and increased tyrosine nitration. We have now studied the subcellular origin of superoxide formation in endothelial cells treated with CsA and the biochemical consequences for the function of mitochondrial enzymes. By using electron spin resonance and endothelial cells lacking functional mitochondria, we showed that superoxide anion is generated in mitochondria. This was associated with an effect of CsA on bioenergetic parameters: increased mitochondrial membrane potential and inhibition of cellular respiration. In addition, CsA inhibited the activity of the mitochondrial enzymes aconitase and manganese superoxide dismutase (MnSOD). The use of murine lung endothelial cells deficient in endothelial nitric oxide synthase (eNOS) and NOS/peroxynitrite inhibitors allowed us to establish that the presence of eNOS and concomitant NO synthesis and peroxynitrite formation were essential for CsA induced nitration and inhibition of MnSOD activity. As the latter has been shown to become inactivated by nitration, we sought to identify this modification by mass spectrometry analysis. We found that CsA induced specific MnSOD tyrosine 34 nitration both in the recombinant protein and in endothelial cells overexpressing MnSOD. We propose that CsA induced endothelial damage may be related to increased mitochondrial superoxide formation and subsequent peroxynitrite-dependent nitroxidative damage, specifically targeting MnSOD. The inactivation of this key antioxidant enzyme by tyrosine nitration represents a pathophysiological cellular mechanism contributing to self-perpetuation and amplification of CsA-related vascular toxicity.Cardiovascular research 07/2010; 87(2):356-65. · 5.80 Impact Factor -
Article: Statistical model to analyze quantitative proteomics data obtained by 18O/16O labeling and linear ion trap mass spectrometry: application to the study of vascular endothelial growth factor-induced angiogenesis in endothelial cells.
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ABSTRACT: Statistical models for the analysis of protein expression changes by stable isotope labeling are still poorly developed, particularly for data obtained by 16O/18O labeling. Besides large scale test experiments to validate the null hypothesis are lacking. Although the study of mechanisms underlying biological actions promoted by vascular endothelial growth factor (VEGF) on endothelial cells is of considerable interest, quantitative proteomics studies on this subject are scarce and have been performed after exposing cells to the factor for long periods of time. In this work we present the largest quantitative proteomics study to date on the short term effects of VEGF on human umbilical vein endothelial cells by 18O/16O labeling. Current statistical models based on normality and variance homogeneity were found unsuitable to describe the null hypothesis in a large scale test experiment performed on these cells, producing false expression changes. A random effects model was developed including four different sources of variance at the spectrum-fitting, scan, peptide, and protein levels. With the new model the number of outliers at scan and peptide levels was negligible in three large scale experiments, and only one false protein expression change was observed in the test experiment among more than 1000 proteins. The new model allowed the detection of significant protein expression changes upon VEGF stimulation for 4 and 8 h. The consistency of the changes observed at 4 h was confirmed by a replica at a smaller scale and further validated by Western blot analysis of some proteins. Most of the observed changes have not been described previously and are consistent with a pattern of protein expression that dynamically changes over time following the evolution of the angiogenic response. With this statistical model the 18O labeling approach emerges as a very promising and robust alternative to perform quantitative proteomics studies at a depth of several thousand proteins.Molecular & Cellular Proteomics 02/2009; 8(5):1130-49. · 7.40 Impact Factor -
Article: Quantitative proteomics of mitochondrial membrane proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis, 16O/18O stable isotope labeling and linear ion trap mass spectrometry
Proteómica: revista de la Sociedad Española de Proteómica, ISSN 1888-0096, 2007, pags. 29-34.
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Institutions
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2011
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Centro De Biología Molecular Severo Ochoa
Madrid, Madrid, Spain
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2009
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Spanish National Centre for Cardiovascular Research
Madrid, Madrid, Spain
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