Rosana Navajas - Academia.edu (original) (raw)
Papers by Rosana Navajas
Molecular & Cellular Proteomics, 2011
The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIA... more The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIAPE (Minimum Information About a Proteomics Experiment) guidelines should improve proteomics data sharing within the scientific community. Proteomics journals have encouraged the use of these standards and guidelines to improve the quality of experimental reporting and ease the evaluation and publication of manuscripts. However, there is an evident lack of bioinformatics tools specifically designed to create and edit standard file formats and reports, or embed them within proteomics workflows. In this article, we describe a new web-based software suite (The ProteoRed MIAPE web toolkit) that performs several complementary roles related to proteomic data standards. First, it can verify that the reports fulfill the minimum information requirements of the corresponding MIAPE modules, highlighting inconsistencies or missing information. Second, the toolkit can convert several XMLbased data standards directly into human readable MIAPE reports stored within the ProteoRed MIAPE repository. Finally, it can also perform the reverse operation, allowing users to export from MIAPE reports into XML files for computational processing, data sharing, or public database submission. The toolkit is thus the first application capable of automatically linking the PSI's MIAPE modules with the corresponding XML data exchange standards, enabling bidirectional conversions. This toolkit is freely available at
Journal of chromatography. B, Biomedical applications, Jan 17, 1995
This paper describes an alternative HPLC method for the determination of testosterone and epitest... more This paper describes an alternative HPLC method for the determination of testosterone and epitestosterone, which is simple, rapid, selective, sensitive and reproducible. Samples were prepared using a previous enzymatic hydrolysis with liquid-liquid extraction. The determination was carried out on a Hypersil BDS-C18 reversed-phase column with gradient elution and UV absorbance detection (240 nm). The limits of quantification (signal-to-noise ratio = 6) were 20 ng/ml for testosterone and 30 ng/ml for epitestosterone.
PLoS ONE, 2014
hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nu... more hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nuclear RNA metabolism and is present in cytoplasmic RNA granules involved in localized translation. Here we have studied whether hCLE shares common interactors in the nucleus and the cytosol, which could shed light on its participation in the sequential phases of RNA metabolism. Nuclear and cytoplasmic purified hCLE-associated factors were identified and proteins involved in mRNA metabolism, motor-related proteins, cytoskeletal and translation-related factors were found. Purified hCLE complexes also contain RNAs and as expected some hCLE-interacting proteins (DDX1, HSPC117, FAM98B) were found both in the nucleus and the cytoplasm. Moreover, endogenous hCLE fractionates in protein complexes together with DDX1, HSPC117 and FAM98B and silencing of hCLE down-regulates their nuclear and cytosolic accumulation levels. Using a photoactivatable hCLE-GFP protein, nuclear import and export of hCLE was observed indicating that hCLE is a shuttling protein. Interestingly, hCLE nuclear import required active transcription, as did the import of DDX1, HSPC117 and FAM98B proteins. The data indicate that hCLE probably as a complex with DDX1, HSPC117 and FAM98B shuttles between the nucleus and the cytoplasm transporting RNAs suggesting that this complex has a prominent role on nuclear and cytoplasmic RNA fate. Citation: Pérez-González A, Pazo A, Navajas R, Ciordia S, Rodriguez-Frandsen A, et al. (2014) hCLE/C14orf166 Associates with DDX1-HSPC117-FAM98B in a Novel Transcription-Dependent Shuttling RNA-Transporting Complex. PLoS ONE 9(3): e90957.
Talanta, 2010
An evaluation of the ICPL (isotope-coded protein labeling) non-isobaric labeling technique was pe... more An evaluation of the ICPL (isotope-coded protein labeling) non-isobaric labeling technique was performed using two different biological models. Two samples containing phage T4 capsids were mixed in a 1:1 ratio after being labeled with the light or heavy versions of the ICPL reagent. The analysis of this proteome demonstrated the feasibility of this approach for differential quantitative proteomics and was employed to optimize the experimental parameters of the ICPL workflow. ICPL-mediated analysis of two more complex proteomes, those of a Salmonella enterica serovar Typhimurium virulent strain and an isogenic attenuated mutant, and its comparison with the results obtained in a 2D-PAGE "classical" approach confirmed that ICPL is a valuable alternative to other labeling techniques currently in use. In addition, our results suggest that labeling at the peptide level instead of following the standard ICPL workflow should increase both the number of proteins quantified and the reliability of the quantification.
Methods in Molecular Biology, 2010
Major difficulties in phosphoprotein analysis relate to the presence of a huge number of nonphosp... more Major difficulties in phosphoprotein analysis relate to the presence of a huge number of nonphosphorylated proteins and to the wide concentration dynamic range among them. In order to overcome the analysis complexity, specific clean-up and highly efficient enrichment procedures are mandatory prior to the chromatographic separation and identification by tandem mass spectrometry. In this chapter, a procedure based on immobilized metal affinity chromatography (IMAC)/reversed-phase phosphopeptide purification and analysis by nanoHPLC-ESI-MS/MS with ion trap is described in detail. CID (collision-induced dissociation) and ETD (electron-transfer dissociation) fragmentation techniques are used in combination to specifically determine phosphorylation sites inside the peptide sequences, through the analysis of MS/MS spectra.
Molecular & Cellular Proteomics, 2008
Tandem mass spectrometry-based proteomics is currently in great demand of computational methods t... more Tandem mass spectrometry-based proteomics is currently in great demand of computational methods that facilitate the elimination of likely false positives in peptide and protein identification. In the last few years, a number of new peptide identification programs have been described, but scores or other significance measures reported by these programs cannot always be directly translated into an easy to interpret error rate measurement such as the false discovery rate. In this work we used generalized lambda distributions to model frequency distributions of database search scores computed by MASCOT, X!TANDEM with k-score plug-in, OMSSA, and InsPecT. From these distributions, we could successfully estimate p values and false discovery rates with high accuracy. From the set of peptide assignments reported by any of these engines, we also defined a generic protein scoring scheme that enabled accurate estimation of protein-level p values by simulation of random score distributions that was also found to yield good estimates of protein-level false discovery rate. The performance of these methods was evaluated by searching four freely available data sets ranging from 40,000 to 285,000 MS/MS spectra.
Molecular & Cellular Proteomics, 2011
The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIA... more The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIAPE (Minimum Information About a Proteomics Experiment) guidelines should improve proteomics data sharing within the scientific community. Proteomics journals have encouraged the use of these standards and guidelines to improve the quality of experimental reporting and ease the evaluation and publication of manuscripts. However, there is an evident lack of bioinformatics tools specifically designed to create and edit standard file formats and reports, or embed them within proteomics workflows. In this article, we describe a new web-based software suite (The ProteoRed MIAPE web toolkit) that performs several complementary roles related to proteomic data standards. First, it can verify that the reports fulfill the minimum information requirements of the corresponding MIAPE modules, highlighting inconsistencies or missing information. Second, the toolkit can convert several XMLbased data standards directly into human readable MIAPE reports stored within the ProteoRed MIAPE repository. Finally, it can also perform the reverse operation, allowing users to export from MIAPE reports into XML files for computational processing, data sharing, or public database submission. The toolkit is thus the first application capable of automatically linking the PSI's MIAPE modules with the corresponding XML data exchange standards, enabling bidirectional conversions. This toolkit is freely available at
Journal of Proteome Research, 2011
The RcsC, RcsD, and RcsB proteins compose a system used by enteric bacteria to sense envelope str... more The RcsC, RcsD, and RcsB proteins compose a system used by enteric bacteria to sense envelope stress. Signal transmission occurs from the sensor RcsC to the transcriptional regulator RcsB. Accessory proteins, such as IgaA, are known to adjust the response level. In a previous transcriptomic study, we uncovered 85 genes differentially expressed in Salmonella enterica serovar Typhimurium igaA mutants. Here, we extended these observations to proteomics by performing differential isotope-coded protein labeling (ICPL) followed by liquid chromatographyÀelectrospray ionization tandem mass spectrometry. Five-hundred five proteins were identified and quantified, with 75 of them displaying significant changes in response to alterations in the RcsCDB system. Divergent expression at the RNA and protein level was observed for the metabolic genes pckA and metE, involved in gluconeogenesis and methionine synthesis, respectively. When analyzed in diverse environmental conditions, including the intracellular niche of eukaryotic cells, inverse regulation was more evident for metE and in bacteria growing in defined minimal medium or to stationary phase. The RcsCDB system was also shown to repress the synthesis of the small RNA FnrS, previously reported to modulate metE expression. Collectively, these findings provide new insights into post-transcriptional regulatory mechanisms involving the RcsCDB system and its control over metabolic functions.
Journal of Chromatography A, 2000
Different reversed-phase columns for basic analytes were compared for the simultaneous determinat... more Different reversed-phase columns for basic analytes were compared for the simultaneous determination of ephedrines in urine, such as LiChrospher 60 RP-Select B, LiChrospher 100 RP18, Hypersil BDS-C18, Inertsil ODS-2, Spherisorb ODS-B and Symmetry Shield RP8. Symmetry Shield was the only column which did not require the use of high concentrations of buffer and triethylamine. With this column, a good separation of the six ephedrines and the internal standard was achieved using 50 mM phosphate buffer-25 mM triethylamine as a mobile phase. Linearity, precision and accuracy were satisfactory for the levels usually found in urine. Due to these all parameters the developed analytical method was found to be suitable for the application in the doping field.
Journal of Biotechnology, 2012
Deficiency in the translocase complex (SecG mutant strain) or in the major type I signal peptidas... more Deficiency in the translocase complex (SecG mutant strain) or in the major type I signal peptidase (SipY mutant strain) function in Streptomyces lividans resulted, as expected, in a drastic reduction of secretory protein production and in a bald phenotype. The transcriptional profiling of both strains showed that the expression of a set of genes involved in the morphological differentiation process was down regulated in both mutant strains (bldG, bldN and bldM), whereas bldA and bldH were only down-regulated in the SipY mutant strain. Consistently, low temperature scanning electron microscopy revealed that the disruption of sipY had a more noticeable effect in the growth/morphological aspect of the mycelium than that of secG, suggesting that in the sipY mutant, the blockage of the export process might have more severe consequences than in the secG mutant. In both cases, the likely degradation of the proteins that cannot be secreted might provide nutrients that might be responsible for the lack of induction of the bald cascade, which is thought to be triggered under conditions of nutritional limitation.
Journal of Proteomics, 2013
Mass spectrometry is already a well-established protein identification tool and recent methodolog... more Mass spectrometry is already a well-established protein identification tool and recent methodological and technological developments have also made possible the extraction of quantitative data of protein abundance in large-scale studies. Several strategies for absolute and relative quantitative proteomics and the statistical assessment of quantifications are possible, each having specific measurements and therefore, different data analysis workflows.
Molecular & Cellular Proteomics, 2011
The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIA... more The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIAPE (Minimum Information About a Proteomics Experiment) guidelines should improve proteomics data sharing within the scientific community. Proteomics journals have encouraged the use of these standards and guidelines to improve the quality of experimental reporting and ease the evaluation and publication of manuscripts. However, there is an evident lack of bioinformatics tools specifically designed to create and edit standard file formats and reports, or embed them within proteomics workflows. In this article, we describe a new web-based software suite (The ProteoRed MIAPE web toolkit) that performs several complementary roles related to proteomic data standards. First, it can verify that the reports fulfill the minimum information requirements of the corresponding MIAPE modules, highlighting inconsistencies or missing information. Second, the toolkit can convert several XMLbased data standards directly into human readable MIAPE reports stored within the ProteoRed MIAPE repository. Finally, it can also perform the reverse operation, allowing users to export from MIAPE reports into XML files for computational processing, data sharing, or public database submission. The toolkit is thus the first application capable of automatically linking the PSI's MIAPE modules with the corresponding XML data exchange standards, enabling bidirectional conversions. This toolkit is freely available at
Journal of chromatography. B, Biomedical applications, Jan 17, 1995
This paper describes an alternative HPLC method for the determination of testosterone and epitest... more This paper describes an alternative HPLC method for the determination of testosterone and epitestosterone, which is simple, rapid, selective, sensitive and reproducible. Samples were prepared using a previous enzymatic hydrolysis with liquid-liquid extraction. The determination was carried out on a Hypersil BDS-C18 reversed-phase column with gradient elution and UV absorbance detection (240 nm). The limits of quantification (signal-to-noise ratio = 6) were 20 ng/ml for testosterone and 30 ng/ml for epitestosterone.
PLoS ONE, 2014
hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nu... more hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nuclear RNA metabolism and is present in cytoplasmic RNA granules involved in localized translation. Here we have studied whether hCLE shares common interactors in the nucleus and the cytosol, which could shed light on its participation in the sequential phases of RNA metabolism. Nuclear and cytoplasmic purified hCLE-associated factors were identified and proteins involved in mRNA metabolism, motor-related proteins, cytoskeletal and translation-related factors were found. Purified hCLE complexes also contain RNAs and as expected some hCLE-interacting proteins (DDX1, HSPC117, FAM98B) were found both in the nucleus and the cytoplasm. Moreover, endogenous hCLE fractionates in protein complexes together with DDX1, HSPC117 and FAM98B and silencing of hCLE down-regulates their nuclear and cytosolic accumulation levels. Using a photoactivatable hCLE-GFP protein, nuclear import and export of hCLE was observed indicating that hCLE is a shuttling protein. Interestingly, hCLE nuclear import required active transcription, as did the import of DDX1, HSPC117 and FAM98B proteins. The data indicate that hCLE probably as a complex with DDX1, HSPC117 and FAM98B shuttles between the nucleus and the cytoplasm transporting RNAs suggesting that this complex has a prominent role on nuclear and cytoplasmic RNA fate. Citation: Pérez-González A, Pazo A, Navajas R, Ciordia S, Rodriguez-Frandsen A, et al. (2014) hCLE/C14orf166 Associates with DDX1-HSPC117-FAM98B in a Novel Transcription-Dependent Shuttling RNA-Transporting Complex. PLoS ONE 9(3): e90957.
Talanta, 2010
An evaluation of the ICPL (isotope-coded protein labeling) non-isobaric labeling technique was pe... more An evaluation of the ICPL (isotope-coded protein labeling) non-isobaric labeling technique was performed using two different biological models. Two samples containing phage T4 capsids were mixed in a 1:1 ratio after being labeled with the light or heavy versions of the ICPL reagent. The analysis of this proteome demonstrated the feasibility of this approach for differential quantitative proteomics and was employed to optimize the experimental parameters of the ICPL workflow. ICPL-mediated analysis of two more complex proteomes, those of a Salmonella enterica serovar Typhimurium virulent strain and an isogenic attenuated mutant, and its comparison with the results obtained in a 2D-PAGE "classical" approach confirmed that ICPL is a valuable alternative to other labeling techniques currently in use. In addition, our results suggest that labeling at the peptide level instead of following the standard ICPL workflow should increase both the number of proteins quantified and the reliability of the quantification.
Methods in Molecular Biology, 2010
Major difficulties in phosphoprotein analysis relate to the presence of a huge number of nonphosp... more Major difficulties in phosphoprotein analysis relate to the presence of a huge number of nonphosphorylated proteins and to the wide concentration dynamic range among them. In order to overcome the analysis complexity, specific clean-up and highly efficient enrichment procedures are mandatory prior to the chromatographic separation and identification by tandem mass spectrometry. In this chapter, a procedure based on immobilized metal affinity chromatography (IMAC)/reversed-phase phosphopeptide purification and analysis by nanoHPLC-ESI-MS/MS with ion trap is described in detail. CID (collision-induced dissociation) and ETD (electron-transfer dissociation) fragmentation techniques are used in combination to specifically determine phosphorylation sites inside the peptide sequences, through the analysis of MS/MS spectra.
Molecular & Cellular Proteomics, 2008
Tandem mass spectrometry-based proteomics is currently in great demand of computational methods t... more Tandem mass spectrometry-based proteomics is currently in great demand of computational methods that facilitate the elimination of likely false positives in peptide and protein identification. In the last few years, a number of new peptide identification programs have been described, but scores or other significance measures reported by these programs cannot always be directly translated into an easy to interpret error rate measurement such as the false discovery rate. In this work we used generalized lambda distributions to model frequency distributions of database search scores computed by MASCOT, X!TANDEM with k-score plug-in, OMSSA, and InsPecT. From these distributions, we could successfully estimate p values and false discovery rates with high accuracy. From the set of peptide assignments reported by any of these engines, we also defined a generic protein scoring scheme that enabled accurate estimation of protein-level p values by simulation of random score distributions that was also found to yield good estimates of protein-level false discovery rate. The performance of these methods was evaluated by searching four freely available data sets ranging from 40,000 to 285,000 MS/MS spectra.
Molecular & Cellular Proteomics, 2011
The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIA... more The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIAPE (Minimum Information About a Proteomics Experiment) guidelines should improve proteomics data sharing within the scientific community. Proteomics journals have encouraged the use of these standards and guidelines to improve the quality of experimental reporting and ease the evaluation and publication of manuscripts. However, there is an evident lack of bioinformatics tools specifically designed to create and edit standard file formats and reports, or embed them within proteomics workflows. In this article, we describe a new web-based software suite (The ProteoRed MIAPE web toolkit) that performs several complementary roles related to proteomic data standards. First, it can verify that the reports fulfill the minimum information requirements of the corresponding MIAPE modules, highlighting inconsistencies or missing information. Second, the toolkit can convert several XMLbased data standards directly into human readable MIAPE reports stored within the ProteoRed MIAPE repository. Finally, it can also perform the reverse operation, allowing users to export from MIAPE reports into XML files for computational processing, data sharing, or public database submission. The toolkit is thus the first application capable of automatically linking the PSI's MIAPE modules with the corresponding XML data exchange standards, enabling bidirectional conversions. This toolkit is freely available at
Journal of Proteome Research, 2011
The RcsC, RcsD, and RcsB proteins compose a system used by enteric bacteria to sense envelope str... more The RcsC, RcsD, and RcsB proteins compose a system used by enteric bacteria to sense envelope stress. Signal transmission occurs from the sensor RcsC to the transcriptional regulator RcsB. Accessory proteins, such as IgaA, are known to adjust the response level. In a previous transcriptomic study, we uncovered 85 genes differentially expressed in Salmonella enterica serovar Typhimurium igaA mutants. Here, we extended these observations to proteomics by performing differential isotope-coded protein labeling (ICPL) followed by liquid chromatographyÀelectrospray ionization tandem mass spectrometry. Five-hundred five proteins were identified and quantified, with 75 of them displaying significant changes in response to alterations in the RcsCDB system. Divergent expression at the RNA and protein level was observed for the metabolic genes pckA and metE, involved in gluconeogenesis and methionine synthesis, respectively. When analyzed in diverse environmental conditions, including the intracellular niche of eukaryotic cells, inverse regulation was more evident for metE and in bacteria growing in defined minimal medium or to stationary phase. The RcsCDB system was also shown to repress the synthesis of the small RNA FnrS, previously reported to modulate metE expression. Collectively, these findings provide new insights into post-transcriptional regulatory mechanisms involving the RcsCDB system and its control over metabolic functions.
Journal of Chromatography A, 2000
Different reversed-phase columns for basic analytes were compared for the simultaneous determinat... more Different reversed-phase columns for basic analytes were compared for the simultaneous determination of ephedrines in urine, such as LiChrospher 60 RP-Select B, LiChrospher 100 RP18, Hypersil BDS-C18, Inertsil ODS-2, Spherisorb ODS-B and Symmetry Shield RP8. Symmetry Shield was the only column which did not require the use of high concentrations of buffer and triethylamine. With this column, a good separation of the six ephedrines and the internal standard was achieved using 50 mM phosphate buffer-25 mM triethylamine as a mobile phase. Linearity, precision and accuracy were satisfactory for the levels usually found in urine. Due to these all parameters the developed analytical method was found to be suitable for the application in the doping field.
Journal of Biotechnology, 2012
Deficiency in the translocase complex (SecG mutant strain) or in the major type I signal peptidas... more Deficiency in the translocase complex (SecG mutant strain) or in the major type I signal peptidase (SipY mutant strain) function in Streptomyces lividans resulted, as expected, in a drastic reduction of secretory protein production and in a bald phenotype. The transcriptional profiling of both strains showed that the expression of a set of genes involved in the morphological differentiation process was down regulated in both mutant strains (bldG, bldN and bldM), whereas bldA and bldH were only down-regulated in the SipY mutant strain. Consistently, low temperature scanning electron microscopy revealed that the disruption of sipY had a more noticeable effect in the growth/morphological aspect of the mycelium than that of secG, suggesting that in the sipY mutant, the blockage of the export process might have more severe consequences than in the secG mutant. In both cases, the likely degradation of the proteins that cannot be secreted might provide nutrients that might be responsible for the lack of induction of the bald cascade, which is thought to be triggered under conditions of nutritional limitation.
Journal of Proteomics, 2013
Mass spectrometry is already a well-established protein identification tool and recent methodolog... more Mass spectrometry is already a well-established protein identification tool and recent methodological and technological developments have also made possible the extraction of quantitative data of protein abundance in large-scale studies. Several strategies for absolute and relative quantitative proteomics and the statistical assessment of quantifications are possible, each having specific measurements and therefore, different data analysis workflows.