Shotgun proteomic analysis of the unicellular alga Ostreococcus tauri

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 highlight 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 illumination, after 24h of darkness and under 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.     

A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus

Background  

Eucalypts are the most widely planted hardwood trees in the world occupying globally more than 18 million hectares as an important source of carbon neutral renewable energy and raw material for pulp, paper and solid wood. Quantitative Trait Loci (QTLs) in Eucalyptus have been localized on pedigree-specific RAPD or AFLP maps seriously limiting the value of such QTL mapping efforts for molecular breeding. The availability of a genus-wide genetic map with transferable microsatellite markers has become a must for the effective advancement of genomic undertakings. This report describes the development of a novel set of 230 EMBRA microsatellites, the construction of the first comprehensive microsatellite-based consensus linkage map for Eucalyptus and the consolidation of existing linkage information for other microsatellites and candidate genes mapped in other species of the genus.     

Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance

The proteomic composition of the Arabidopsis (Arabidopsis thaliana) Golgi apparatus is currently reasonably well documented; however, little is known about the relative abundances between different proteins within this compartment. Accurate quantitative information of Golgi resident proteins is of great importance: it facilitates a better understanding of the biochemical processes that take place within this organelle, especially those of different polysaccharide synthesis pathways. Golgi resident proteins are challenging to quantify because the abundance of this organelle is relatively low within the cell. In this study, an organelle fractionation approach targeting the Golgi apparatus was combined with a label-free quantitative mass spectrometry (data-independent acquisition method using ion mobility separation known as LC-IMS-MS^E [or HDMS^E]) to simultaneously localize proteins to the Golgi apparatus and assess their relative quantity. In total, 102 Golgi-localized proteins were quantified. These data show that organelle fractionation in conjunction with label-free quantitative mass spectrometry is a powerful and relatively simple tool to access protein organelle localization and their relative abundances. The findings presented open a unique view on the organization of the plant Golgi apparatus, leading toward unique hypotheses centered on the biochemical processes of this organelle.The plant Golgi apparatus plays an important role in protein and lipid glycosylation and sorting as well as biosynthesis of large amounts of extracellular polysaccharides. It contains a large and diverse set of glycosyltransferases and other enzymes that are required for the synthesis and modification of these polysaccharides (Parsons et al., 2012b; Oikawa et al., 2013). The protein composition of this organelle has been the focus of a number of studies; however, these studies largely report a catalog of Golgi-localized proteins, and to date, there are no comprehensive data on the relative abundance of the different protein constituents of the Golgi apparatus (Dunkley et al., 2004, 2006; Sadowski et al., 2008; Nikolovski et al., 2012; Groen et al., 2014). The quantification of the plant Golgi proteome has been considered challenging, because this organelle is proportionally of low abundance in the cell; therefore, its constituent proteins are rarely identified in conventional proteomics experiments. Investigation of such low-abundance proteins generally requires sample fractionation on the organelle, protein, or peptide level (Stasyk and Huber, 2004; Haynes and Roberts, 2007; Di Palma et al., 2012).Here, an organelle fractionation approach in conjunction with label-free quantitative proteomic analysis was used to assess the localization and relative abundance of proteins within the plant Golgi apparatus. Label-free quantification is an increasingly popular alternative to isotopic tagging quantitative methods; it does not require labeling reagents and can be applied to an unlimited number of samples (Neilson et al., 2011; Evans et al., 2012). This is particularly appealing within plant proteomics, because the most conventional labeling strategy, Stable Isotope Labeling by Amino Acids in Cell Culture, is not easily suited for quantitative plant proteomic studies. The average labeling efficiency achieved using exogenous amino acid supply to Arabidopsis (Arabidopsis thaliana) cell cultures was found to be only 70% to 80% (Gruhler et al., 2005). Quantitative strategies with ¹⁵N metabolic labeling have been described for plant proteome analysis; however, care should be taken to ensure complete ¹⁵N incorporation, because even small amounts of ¹⁴N in the labeled sample can have significant detrimental effects on the number of peptide identifications (Nelson et al., 2007; Guo and Li, 2011; Arsova et al., 2012).In all label-free methods, samples under comparison are analyzed during separate mass spectrometry (MS) experiments (Neilson et al., 2011). The information from identified peptides is then used for relative and/or absolute quantification. The simplest label-free method involves taking the number of spectra acquired and assigned to peptides from the same protein as a measure of abundance (Ishihama et al., 2005). In an alternative approach, ion current recorded for a peptide ion is used as a measure of its abundance. The assumption is made that ion intensity is proportional to peptide amount in the sample analyzed, which holds true for nanoflow and microflow liquid chromatography (LC) systems (Levin et al., 2011; Christianson et al., 2013). Comparing peptide ion current between samples is, thus, widely used for relative quantification (Silva et al., 2005). To allow such comparison, a peptide must be identified across all samples under investigation, which is often challenging in LC-MS experiments given the highly complex nature of proteomics samples that contain tens of thousands of different peptides (Michalski et al., 2011). Hence, most relative ion intensity-based label-free approaches usually involve a step of identification transfer (Pasa-Tolíc et al., 2004). This involves matching ions from different acquisitions (in one of which, the ion has not been identified and is assigned the sequence from its matching pair in the other acquisition).Additionally, label-free proteomics can be used for absolute quantification (i.e. to estimate abundance of different proteins relative to each other within a given sample). Several different approaches have been suggested on how to convert peptide intensities to protein amounts (for comparison, see Wilhelm et al., 2014). One of the first such methods was Top-3 described by Silva et al. (2006b), who made a notable and unexpected observation, stating that the average MS signal response for the three most abundant peptides per 1 mol of protein is constant within a coefficient of variation of less than 10% (Silva et al., 2006b).In all these approaches, the peptide ion current is typically computed as the area under the curve of the chromatographic elution profile that is reconstituted from separate MS1 survey scans in which intact precursors are recorded. Determining a chromatographic profile accurately requires that the MS1 scans are performed at optimal frequency (Lange et al., 2008) and for optimal duration to record the MS1 signal at a high signal-to-noise ratio. In typical data-dependent acquisitions, however, the mass spectrometer oscillates between MS1 survey scans recording the mass/charge (m/z) for precursor peptide ions and then, a series of MS2 scans fragmenting one peptide ion precursor at a time, producing fragmentation spectra necessary for identification (Sadygov et al., 2004). As a result, the duration and frequency of MS2 scans determine the identification rate in data-dependent acquisition experiments but compromise time spent in MS1 required for accurate area under the curve quantification. Several groups have suggested data-independent acquisition, in which individual peptide ions are not selected for fragmentation but rather, groups of peptides of similar m/z are fragmented together. The exact number of cofragmented precursors depends on the speed and sensitivity of instrument configuration (for review, see Law and Lim, 2013). The simplest approach involves alternating between low-energy and high-energy scans of equal duration; low-energy scans record precursor peptide ions, whereas in high-energy scans, all precursors entering the mass spectrometer are cofragmented, and their fragments are recorded simultaneously. The method was called MS^E for Waters qTOF Mass Spectrometers (Geromanos et al., 2009) or all-ion fragmentation for Thermo Orbitrap Mass Spectrometers (Geiger et al., 2010). The analysis required downstream of this type of data acquisition is challenging given that the information of fragment origin (i.e. from what precursor peptide ion fragment was generated) is lost completely and that the high number of coeluting peptides is expected to create highly overlapping fragment spectra on fragmentation. To address this problem, Hoaglund-Hyzer and Clemmer (2001) have suggested fractionating peptides by ion mobility separation before fragmentation and MS and assigning fragments to precursors based on similarity of both chromatographic and mobility profiles (Hoaglund-Hyzer and Clemmer, 2001). The method was termed parallel fragmentation, and since that time, it has been commercialized by Waters as IMS-MS^E or HDMS^E (Shliaha et al., 2013).To date, the application of label-free quantitative proteomics to plant biology has been very limited. Recently, Helm et al. (2014) applied the LC-IMS-MS^E with Top-3 quantification to quantify the Arabidopsis chloroplast stroma proteome, allowing quantitative modeling of chloroplast metabolism. Two other works used the LC-MS^E method to assess the quantitative changes of cytosolic ribosomal proteins in response to Suc feeding and the extracellular proteome in response to salicylic acid (Cheng et al., 2009; Hummel et al., 2012).A number of proteomics approaches have been described to assess protein localization on a large scale (for review, see Gatto et al., 2010). Purification approaches attempt to isolate organelles to high levels of purity and subsequently identify and quantify proteins using LC-MS; however, such attempts yield limiting success and high false discovery rates (Andersen et al., 2002; Parsons et al., 2012a). A known limitation of this technique is the inability to completely isolate an organelle of interest, which combined with high proteome dynamic range, can result in some more abundant contaminants being identified and quantified at higher amounts than the target organelle residents. Moreover, even if a target organelle could be isolated to a certain degree of purity, it would still be impossible to deconvolute organelle residents from transient proteins that traffic through the target organelle. This becomes especially challenging for the organelles of the secretory pathway. To address these challenges, several groups applied fractionation of all organelles by gradient centrifugation and subsequent protein quantification by LC-MS. This produces distributions across the gradient for all quantified proteins, which are then used to assign organelle localization based on the specific distributions of organelle marker proteins. This effectively solves the problem of organelle contamination and protein trafficking, because a protein is expected to have a distribution characteristic of its organelle of residence, even if it is identified in all fractions, including those enriched in other organelles. Current variations of this method differ mostly by the LC-MS strategy used for quantification; for example, spectral counting was applied for protein-correlating profiles (Andersen et al., 2003), isobaric mass tagging (Nikolovski et al., 2012) and isotope-coded affinity tagging (Dunkley et al., 2004) were applied for localization of organelle proteins by isotope tagging (LOPIT), and Stable Isotope Labeling by Amino Acids in Cell Culture was applied for nucleolus/nucleus/cytosolic fractionation (Boisvert and Lamond, 2010).Here, a label-free LC-IMS-MS^E method was used for the analysis of density ultracentrifugation fractions enriched for the Golgi apparatus. First, we use relative label-free quantification involving identification transfer using the previously published synapter algorithm (Bond et al., 2013) to assess distributions of Golgi-localized proteins across the density gradient. These distributions are significantly different from those of residents of other organelles, which results in unambiguous protein assignment to the Golgi apparatus by multivariate data analysis. Second, the Top-3 absolute quantification method as implemented in Protein Lynx Global Server (PLGS) was used to rank order the Golgi-localized proteins by abundance in the fraction most enriched for Golgi apparatus. In conclusion, we present the analysis of protein distribution and abundances of the Golgi apparatus-enriched portion of the ultracentrifugation density gradient, allowing for simultaneous protein quantification and localization and leading to the assessment of relative abundances of 102 Golgi-localized proteins.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Comparative analysis of the zeta-crystallin/quinone reductase gene in guinea pig and mouse

zeta-Crystallin is a novel nicotinamide adenine dinucleotide phosphate:quinone reductase, present at enzymatic levels in various tissues of different species, which is highly expressed in the lens of some hystricomorph rodents and camelids. We report here the complementary DNA (cDNA) cloning of zeta-crystallin from liver libraries in guinea pig (Cavia porcellus), where zeta-crystallin is highly expressed in the lens, and in the laboratory mouse (Mus musculus), where expression in the lens occurs only at enzymatic levels. A 5' untranslated sequence different from the one previously reported for the guinea pig lens cDNA was found in these clones. We also report the isolation of genomic clones including the complete guinea pig zeta-crystallin gene and the 5' region of this gene in mouse. These results show the presence of two promoters in the guinea pig zeta-crystallin gene, one responsible for expression at enzymatic levels and the other responsible for the high expression in the lens. The guinea pig lens promoter is not present in the mouse gene. This is the first example in which the recruitment of an enzyme as a lens crystallin can be explained by the acquisition of an alternative lens- specific promoter.      

Quantification of the magnification and distortion effects of a pediatric flexible video-bronchoscope

Background

Flexible video bronchoscopes, in particular the Olympus BF Type 3C160, are commonly used in pediatric respiratory medicine. There is no data on the magnification and distortion effects of these bronchoscopes yet important clinical decisions are made from the images. The aim of this study was to systematically describe the magnification and distortion of flexible bronchoscope images taken at various distances from the object.

Methods

Using images of known objects and processing these by digital video and computer programs both magnification and distortion scales were derived.

Results

Magnification changes as a linear function between 100 mm (×1) and 10 mm (×9.55) and then as an exponential function between 10 mm and 3 mm (×40) from the object. Magnification depends on the axis of orientation of the object to the optic axis or geometrical axis of the bronchoscope. Magnification also varies across the field of view with the central magnification being 39% greater than at the periphery of the field of view at 15 mm from the object. However, in the paediatric situation the diameter of the orifices is usually less than 10 mm and thus this limits the exposure to these peripheral limits of magnification reduction. Intraclass correlations for measurements and repeatability studies between instruments are very high, r = 0.96. Distortion occurs as both barrel and geometric types but both types are heterogeneous across the field of view. Distortion of geometric type ranges up to 30% at 3 mm from the object but may be as low as 5% depending on the position of the object in relation to the optic axis.

Conclusion

We conclude that the optimal working distance range is between 40 and 10 mm from the object. However the clinician should be cognisant of both variations in magnification and distortion in clinical judgements.     

Evidence for independent recruitment of zeta-crystallin/quinone reductase (CRYZ) as a crystallin in camelids and hystricomorph rodents

Zeta-crystallin/quinone reductase (CRYZ) is an NADPH oxidoreductase expressed at very high levels in the lenses of two groups of mammals: camelids and some hystricomorph rodents. It is also expressed at very low levels in all other species tested. Comparative analysis of the mechanisms mediating the high expression of this enzyme/crystallin in the lens of the Ilama (Lama guanacoe) and the guinea pig (Cavia porcellus) provided evidence for independent recruitment of this enzyme as a lens crystallin in both species and allowed us to elucidate for the first time the mechanism of lens recruitment of an enzyme- crystallin. The data presented here show that in both species such recruitment most likely occurred through the generation of new lens promoters from nonfunctional intron sequences by the accumulation of point mutations and/or small deletions and insertions. These results further support the idea that recruitment of CRYZ resulted from an adaptive process in which the high expression of CRYZ in the lens provides some selective advantage rather than from a purely neutral evolutionary process.      

Transcriptomic profile of host response in Japanese encephalitis virus infection

Background

The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a Trypanosoma cruzi CD8⁺ T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8⁺ T cell epitope was cloned and expressed based on two different genomic insertion sites: in the fg loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope.

Results

Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by T. cruzi with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8⁺ T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of T. cruzi.

Conclusions

We conclude that the YF 17D platform is useful to express T. cruzi (Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model T. cruzi epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI epitope, will elicit better T-CD8+ responses to the latter. It is likely that additional antigens and recombinant virus formulations will be necessary to generate a protective response.     

Complex-dependent histone acetyltransferase activity of KAT8 determines its role in transcription and cellular homeostasis

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