Protein synthesis and degradation determine the cellular levels of proteins, and their control he... more Protein synthesis and degradation determine the cellular levels of proteins, and their control hence enables organisms to respond to environmental change. Experimentally, these are little known proteome parameters, however recently, SILAC-based mass spectrometry studies have begun to quantify turnover in the proteomes of cell lines, yeast and animals. Here, we present a proteome-scale method to quantify turnover and calculate synthesis and degradation rate constants of individual proteins in autotrophic organisms such as algae and plants. The workflow is based on the automated analysis of partial stable isotope incorporation with <sup>15</sup>N. We applied it in a study of the unicellular pico-alga <i>Ostreococcus tauri</i> and observed high relative turnover in chloroplast-encoded ATPases (0.42%-0.58% h<sup>-1</sup>), core photosystem II proteins (0.34%-0.51% h<sup>-1</sup>) and RbcL (0.47% h<sup>-1</sup>), while nuclear-encoded RbcS2 is more stable (0.23% h<sup>-1</sup>). Mitochondrial targeted ATPases (0.14%-0.16% h<sup>-1</sup>), photosystem antennae (0.09%-0.14% h<sup>-1</sup>) and histones (0.07%-0.1% h<sup>-1</sup>) were comparatively stable. The calculation of degradation and synthesis rate constants k<sub>deg</sub> and k<sub>syn</sub> confirms RbcL as the bulk contributor to overall protein turnover. This study performed over 144h of incorporation reveals dynamics of protein complex subunits as well as isoforms targeted to different organelles.
Apolipoprotein E (APOE)-ɛ4 is associated with a deleterious outcome after ischemic brain injury, ... more Apolipoprotein E (APOE)-ɛ4 is associated with a deleterious outcome after ischemic brain injury, which may involve abnormal regulation of mitochondrial function. We have assessed the mitochondrial proteomic response of APOE-ɛ3 and APOE-ɛ4 transgenic mice to transient global ischemic injury in the hippocampus. A genotype-dependent increase in ApoE levels in mitochondria was observed after ischemia, with APOE-ɛ4 mice showing significantly greater increases than APOE-ɛ3 mice. Quantitative analysis of the mitochondria-enriched fractions was performed using liquid-chromatography mass spectrometry coupled to label-free analysis. Of the 1,067 identified proteins, 274 were mitochondria associated. Mitochondrial protein expression was significantly different between genotypes under basal conditions as well as in response to global ischemia. A total of 12 mitochondrial proteins (including respiratory chain proteins NDUFA11, NDUFS3, NDUF5B, ATP5J, as well as ETFA, CYB5B, ATP6V1A, HSPA1B, OXR1, GLUL, IARS2, and PHYHIPL) were significantly altered with respect to genotype, global ischemia, or their interaction (P<0.01). A compelling interactome, created using proteins found to be significantly modulated by global ischemia (P<0.05), involved proteins that regulate energy production and oxidative stress. Thus, APOE genotype has a differential effect on the mitochondrial protein expression in the absence and presence of an injury, which may underlie the differing genotype susc
Ostreococcus tauri is a unicellular green alga and amongst the smallest and simplest free-living ... more Ostreococcus tauri is a unicellular green alga and amongst the smallest and simplest free-living eukaryotes. The O. tauri genome sequence was determined in 2006. Molecular, physiological and taxonomic data that has been generated since then highlights its potential as a simple model species for algae and plants. However, its proteome remains largely unexplored. This paper describes the global proteomic study of O. tauri, using mass spectrometry-based approaches: phosphopeptide enrichment, cellular fractionation, label-free quantification and (15)N metabolic labeling. The O. tauri proteome was analyzed under the following conditions: sampling at different times during the circadian cycle, after 24h of daylight, after 24h of darkness and various nitrogen source supply levels. Cell cycle related proteins such as dynamin and kinesin were significantly up-regulated during the daylight-to-darkness transition. This is reflected by their higher intensity at ZT13 and this transition phase coincides with the end of mitosis. Proteins involved in several metabolic mechanisms were found to be up-regulated under low nitrogen conditions, including carbon storage pathways, glycolysis, phosphate transport, and the synthesis of inorganic polyphosphates. Ostreococcus tauri responds to low nitrogen conditions by reducing its nitrogen assimilation machinery which suggests an atypical adaptation mechanism for coping with a nutrient-limited environment.
Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stabil... more Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro fluorescence-resonance-energy-transfer-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-specific C-terminal hydrolase activity. This assay has applications in SUMO protease characterization, enzyme kinetic analysis, determination of SUMO protease activity in eukaryotic cell extracts, and high-throughput inhibitor screening. In addition, while demonstrating such uses, we show that the SUMO-1 processing activity in crude HeLa cell extracts is far greater than that of SUMO-2, implying that differential maturation rates of SUMO paralogs in vivo may be functionally significant. The high degree of structural conservation across the ubiquitin-like protein superfamily suggests that the general principle of this assay should be applicable to other post-translational protein modification systems.
The clinical diagnosis of cervical neoplasia by spectroscopic methods is potentially a reliable, ... more The clinical diagnosis of cervical neoplasia by spectroscopic methods is potentially a reliable, fast and cost-effective alternative to the conventional smear test. However, it is currently limited by significant inter-patient variation in the spectroscopic properties of the cervix. Characterisation of suitable in vitro models of the spectroscopic changes that take place during neoplastic progression may prove to be a significant step towards the successful development of reliable in vivo systems. In this study, we used organotypic epithelial raft culture as an in vitro model of cervical tissue to analyse changes in the fluorescence properties of surface squamous epithelium that are associated with the development of neoplastic disease. Collagen plugs lined by primary human keratinocytes (PHKs) were used to model the normal cervical epithelium, and plugs lined by cells of the SiHa line were used as a model of neoplastic cervical tissue. Fluorescence emission spectra of these rafts were recorded at excitation wavelengths in the 250-330 nm range, complementing previous work published at longer wavelengths. Normalised, truncated emission spectra were analysed using multivariate principal component analysis. We successfully distinguished between in vitro models of normal and neoplastic cervical tissue and demonstrated a differential effect of acetic acid, which enhances the discrimination of normal from neoplastic tissue. Identification of these differences between in vitro organotypic epithelial rafts may ultimately aid the discrimination of cervical lesions in vivo.
Protein–protein binding and signaling pathways are important fields of biomedical science. Here w... more Protein–protein binding and signaling pathways are important fields of biomedical science. Here we report simple optical methods for the determination of the equilibrium binding constant Kd of protein–protein interactions as well as quantitative studies of biochemical cascades. The techniques are based on steady-state and time-resolved fluorescence resonance energy transfer (FRET) between ECFP and Venus-YFP fused to proteins of the SUMO family. Using FRET has several advantages over conventional free-solution techniques such as isothermal titration calorimetry (ITC): Concentrations are determined accurately by absorbance, highly sensitive binding signals enable the analysis of small quantities, and assays are compatible with multi-well plate format. Most importantly, our FRET-based techniques enable us to measure the effect of other molecules on the binding of two proteins of interest, which is not straightforward with other approaches. These assays provide powerful tools for the study of competitive biochemical cascades and the extent to which drug candidates modify protein interactions.
We present the application of a targeted liquid chromatography/mass spectrometry (LC/MS) approach... more We present the application of a targeted liquid chromatography/mass spectrometry (LC/MS) approach developed on a linear ion trap for the evaluation of the abundance of cytoplasmic proteins from a HeLa cell extract. Using a standard data-dependent approach, we identified some specific peptides from this extract which were also commercially available in their AQUA form (use for absolute quantitation). For some of the peptides, we observed a non-linear response between the intensity and the added quantity which was then fitted using a quadratic fit. All AQUA peptides spiked into a mix of 3 microg of the HeLa cell digest extract were detected down to 16 fmol. We placed an emphasis on peptide detection which, in this study, is performed using a combination of properties such as three specific Q3-like ion signatures (for a given Q1-like selection) and co-elution with the AQUA peptide counterparts. Detecting a peptide without necessarily identifying it using a search engine imposes less constraint in terms of tandem mass (MS/MS) spectra purity. An example is shown where a peptide is detected using those criteria but could not be identified by Mascot due to its lower abundance. To complement this observation, we used a cross-correlation analysis approach in order to separate two populations of MS/MS fragments based on differences in their elution patterns. Such an approach opens the door to new strategies to analyse lower intensity peptide fragments. An in silico analysis of the human trypsinosome allows the evaluation of how unique are the sets of features that we are using for peptide detection.
Protein synthesis and degradation determine the cellular levels of proteins, and their control he... more Protein synthesis and degradation determine the cellular levels of proteins, and their control hence enables organisms to respond to environmental change. Experimentally, these are little known proteome parameters, however recently, SILAC-based mass spectrometry studies have begun to quantify turnover in the proteomes of cell lines, yeast and animals. Here, we present a proteome-scale method to quantify turnover and calculate synthesis and degradation rate constants of individual proteins in autotrophic organisms such as algae and plants. The workflow is based on the automated analysis of partial stable isotope incorporation with <sup>15</sup>N. We applied it in a study of the unicellular pico-alga <i>Ostreococcus tauri</i> and observed high relative turnover in chloroplast-encoded ATPases (0.42%-0.58% h<sup>-1</sup>), core photosystem II proteins (0.34%-0.51% h<sup>-1</sup>) and RbcL (0.47% h<sup>-1</sup>), while nuclear-encoded RbcS2 is more stable (0.23% h<sup>-1</sup>). Mitochondrial targeted ATPases (0.14%-0.16% h<sup>-1</sup>), photosystem antennae (0.09%-0.14% h<sup>-1</sup>) and histones (0.07%-0.1% h<sup>-1</sup>) were comparatively stable. The calculation of degradation and synthesis rate constants k<sub>deg</sub> and k<sub>syn</sub> confirms RbcL as the bulk contributor to overall protein turnover. This study performed over 144h of incorporation reveals dynamics of protein complex subunits as well as isoforms targeted to different organelles.
Apolipoprotein E (APOE)-ɛ4 is associated with a deleterious outcome after ischemic brain injury, ... more Apolipoprotein E (APOE)-ɛ4 is associated with a deleterious outcome after ischemic brain injury, which may involve abnormal regulation of mitochondrial function. We have assessed the mitochondrial proteomic response of APOE-ɛ3 and APOE-ɛ4 transgenic mice to transient global ischemic injury in the hippocampus. A genotype-dependent increase in ApoE levels in mitochondria was observed after ischemia, with APOE-ɛ4 mice showing significantly greater increases than APOE-ɛ3 mice. Quantitative analysis of the mitochondria-enriched fractions was performed using liquid-chromatography mass spectrometry coupled to label-free analysis. Of the 1,067 identified proteins, 274 were mitochondria associated. Mitochondrial protein expression was significantly different between genotypes under basal conditions as well as in response to global ischemia. A total of 12 mitochondrial proteins (including respiratory chain proteins NDUFA11, NDUFS3, NDUF5B, ATP5J, as well as ETFA, CYB5B, ATP6V1A, HSPA1B, OXR1, GLUL, IARS2, and PHYHIPL) were significantly altered with respect to genotype, global ischemia, or their interaction (P<0.01). A compelling interactome, created using proteins found to be significantly modulated by global ischemia (P<0.05), involved proteins that regulate energy production and oxidative stress. Thus, APOE genotype has a differential effect on the mitochondrial protein expression in the absence and presence of an injury, which may underlie the differing genotype susc
Ostreococcus tauri is a unicellular green alga and amongst the smallest and simplest free-living ... more Ostreococcus tauri is a unicellular green alga and amongst the smallest and simplest free-living eukaryotes. The O. tauri genome sequence was determined in 2006. Molecular, physiological and taxonomic data that has been generated since then highlights its potential as a simple model species for algae and plants. However, its proteome remains largely unexplored. This paper describes the global proteomic study of O. tauri, using mass spectrometry-based approaches: phosphopeptide enrichment, cellular fractionation, label-free quantification and (15)N metabolic labeling. The O. tauri proteome was analyzed under the following conditions: sampling at different times during the circadian cycle, after 24h of daylight, after 24h of darkness and various nitrogen source supply levels. Cell cycle related proteins such as dynamin and kinesin were significantly up-regulated during the daylight-to-darkness transition. This is reflected by their higher intensity at ZT13 and this transition phase coincides with the end of mitosis. Proteins involved in several metabolic mechanisms were found to be up-regulated under low nitrogen conditions, including carbon storage pathways, glycolysis, phosphate transport, and the synthesis of inorganic polyphosphates. Ostreococcus tauri responds to low nitrogen conditions by reducing its nitrogen assimilation machinery which suggests an atypical adaptation mechanism for coping with a nutrient-limited environment.
Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stabil... more Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro fluorescence-resonance-energy-transfer-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-specific C-terminal hydrolase activity. This assay has applications in SUMO protease characterization, enzyme kinetic analysis, determination of SUMO protease activity in eukaryotic cell extracts, and high-throughput inhibitor screening. In addition, while demonstrating such uses, we show that the SUMO-1 processing activity in crude HeLa cell extracts is far greater than that of SUMO-2, implying that differential maturation rates of SUMO paralogs in vivo may be functionally significant. The high degree of structural conservation across the ubiquitin-like protein superfamily suggests that the general principle of this assay should be applicable to other post-translational protein modification systems.
The clinical diagnosis of cervical neoplasia by spectroscopic methods is potentially a reliable, ... more The clinical diagnosis of cervical neoplasia by spectroscopic methods is potentially a reliable, fast and cost-effective alternative to the conventional smear test. However, it is currently limited by significant inter-patient variation in the spectroscopic properties of the cervix. Characterisation of suitable in vitro models of the spectroscopic changes that take place during neoplastic progression may prove to be a significant step towards the successful development of reliable in vivo systems. In this study, we used organotypic epithelial raft culture as an in vitro model of cervical tissue to analyse changes in the fluorescence properties of surface squamous epithelium that are associated with the development of neoplastic disease. Collagen plugs lined by primary human keratinocytes (PHKs) were used to model the normal cervical epithelium, and plugs lined by cells of the SiHa line were used as a model of neoplastic cervical tissue. Fluorescence emission spectra of these rafts were recorded at excitation wavelengths in the 250-330 nm range, complementing previous work published at longer wavelengths. Normalised, truncated emission spectra were analysed using multivariate principal component analysis. We successfully distinguished between in vitro models of normal and neoplastic cervical tissue and demonstrated a differential effect of acetic acid, which enhances the discrimination of normal from neoplastic tissue. Identification of these differences between in vitro organotypic epithelial rafts may ultimately aid the discrimination of cervical lesions in vivo.
Protein–protein binding and signaling pathways are important fields of biomedical science. Here w... more Protein–protein binding and signaling pathways are important fields of biomedical science. Here we report simple optical methods for the determination of the equilibrium binding constant Kd of protein–protein interactions as well as quantitative studies of biochemical cascades. The techniques are based on steady-state and time-resolved fluorescence resonance energy transfer (FRET) between ECFP and Venus-YFP fused to proteins of the SUMO family. Using FRET has several advantages over conventional free-solution techniques such as isothermal titration calorimetry (ITC): Concentrations are determined accurately by absorbance, highly sensitive binding signals enable the analysis of small quantities, and assays are compatible with multi-well plate format. Most importantly, our FRET-based techniques enable us to measure the effect of other molecules on the binding of two proteins of interest, which is not straightforward with other approaches. These assays provide powerful tools for the study of competitive biochemical cascades and the extent to which drug candidates modify protein interactions.
We present the application of a targeted liquid chromatography/mass spectrometry (LC/MS) approach... more We present the application of a targeted liquid chromatography/mass spectrometry (LC/MS) approach developed on a linear ion trap for the evaluation of the abundance of cytoplasmic proteins from a HeLa cell extract. Using a standard data-dependent approach, we identified some specific peptides from this extract which were also commercially available in their AQUA form (use for absolute quantitation). For some of the peptides, we observed a non-linear response between the intensity and the added quantity which was then fitted using a quadratic fit. All AQUA peptides spiked into a mix of 3 microg of the HeLa cell digest extract were detected down to 16 fmol. We placed an emphasis on peptide detection which, in this study, is performed using a combination of properties such as three specific Q3-like ion signatures (for a given Q1-like selection) and co-elution with the AQUA peptide counterparts. Detecting a peptide without necessarily identifying it using a search engine imposes less constraint in terms of tandem mass (MS/MS) spectra purity. An example is shown where a peptide is detected using those criteria but could not be identified by Mascot due to its lower abundance. To complement this observation, we used a cross-correlation analysis approach in order to separate two populations of MS/MS fragments based on differences in their elution patterns. Such an approach opens the door to new strategies to analyse lower intensity peptide fragments. An in silico analysis of the human trypsinosome allows the evaluation of how unique are the sets of features that we are using for peptide detection.
Uploads
Papers by Sarah Martin