PCA2GO: a new multivariate statistics based method to identify highly expressed GO-Terms

<p>Abstract</p> <p>Background</p> <p>Several tools have been developed to explore and search Gene Ontology (GO) databases allowing efficient GO enrichment analysis and GO tree visualization. Nevertheless, identification of highly specific GO-terms in complex data sets is relatively complicated and the display of GO term assignments and GO enrichment analysis by simple tables or pie charts is not optimal. Valuable information such as the hierarchical position of a single GO term within the GO tree (topological ordering), or enrichment within a complex set of biological experiments is not displayed. Pie charts based on GO tree levels are, themselves, one-dimensional graphs, which cannot properly or efficiently represent the hierarchical specificity for the biological system being studied.</p> <p>Results</p> <p>Here we present a new method, which we name PCA2GO, capable of GO analysis using complex multidimensional experimental settings. We employed principal component analysis (PCA) and developed a new score, which takes into account the relative frequency of certain GO terms and their specificity (hierarchical position) within the GO graph. We evaluated the correlation between our representation score <it>R </it>and a standard measure of enrichment, namely <it>p</it>-values to convey the versatility of our approach to other methods and point out differences between our method and commonly used enrichment analyses. Although <it>p </it>values and the <it>R </it>score formally measure different quantities they should be correlated, because relative frequencies of GO terms occurrences within a dataset are an indirect measure of protein numbers related to this term. Therefore they are also related to enrichment. We showed that our score enables us to identify more specific GO-terms i.e. those positioned further down the GO-graph than other common tools used for this purpose. PCA2GO allows visualization and detection of multidimensional dependencies both within the acyclic graph (GO tree) and the experimental settings. Our method is intended for the analysis of several experimental sets, not for one set, like standard enrichment tools. To demonstrate the usefulness of our approach we performed a PCA2GO analysis of a fractionated cardiomyocyte protein dataset, which was identified by enhanced liquid chromatography-mass spectrometry (GeLC-MS). The analysis enabled us to detect distinct groups of proteins, which accurately reflect properties of biochemical cell fractions.</p> <p>Conclusions</p> <p>We conclude that PCA2GO is an alternative efficient GO analysis tool with unique features for detection and visualization of multidimensional dependencies within the dataset under study. PCA2GO reveals strongly correlated GO terms within the experimental setting (in this case different fractions) by PCA group formation and improves detection of more specific GO terms within experiment dependent GO term groups than standard <it>p </it>value calculations.</p

High-Throughput Proteomics Identifies Proteins With Importance to Postantibiotic Recovery in Depolarized Persister Cells

Bacterial populations produce phenotypic variants called persisters to survive harmful conditions. Persisters are highly tolerant to antibiotics and repopulate environments after the stress has vanished. In order to resume growth, persisters have to recover from the persistent state, but the processes behind recovery remain mostly elusive. Deciphering these processes is an essential step toward understanding the persister phenomenon in its entirety. High-throughput proteomics by mass spectrometry is a valuable tool to assess persister physiology during any stage of the persister life cycle, and is expected to considerably contribute to our understanding of the recovery process. In the present study, an Escherichia coli strain, that overproduces the membrane-depolarizing toxin TisB, was established as a model for persistence by the use of high-throughput proteomics. Labeling of TisB persisters with stable isotope-containing amino acids (pulsed-SILAC) revealed an active translational response to ampicillin, including several RpoS-dependent proteins. Subsequent investigation of the persister proteome during postantibiotic recovery by label-free quantitative proteomics identified proteins with importance to the recovery process. Among them, AhpF, a component of alkyl hydroperoxide reductase, and the outer membrane porin OmpF were found to affect the persistence time of TisB persisters. Assessing the role of AhpF and OmpF in TisB-independent persisters demonstrated that the importance of a particular protein for the recovery process strongly depends on the physiological condition of a persister cell. Our study provides important insights into persister physiology and the processes behind recovery of depolarized cells

Neuroproteomics Tools in Clinical Practice

Abstract Neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) are characterized by neuronal impairment that leads to disease-specific changes in the neuronal proteins. The early diagnosis of these disorders is difficult, thus, the need for identifying, developing and using valid clinically applicable biomarkers that meet the criteria of precision, specificity and repeatability is very vital. The application of rapidly emerging technology such as mass spectrometry (MS) in proteomics has opened new avenues to accelerate biomarker discovery, both for diagnostic as well as for prognostic purposes. This review summarizes the most recent advances in the mass spectrometry-based neuroproteomics and analyses the current and future directions in the biomarker discovery for the neurodegenerative diseases.11 This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology

Proteomic analysis of the aqueous humour in eyes with pseudoexfoliation syndrome

Background/aims Pseudoexfoliation syndrome (PEX) is characterised by the production and accumulation of extracellular fibrillar material in the anterior segment of the eye. The pathogenesis of PEX is multifactorial with genetic factors and ageing as contributing factors. Previously, an increased concentration of beta-crystalline B2 (CRYBB2) was observed in the aqueous humour (AH) in eyes with PEX in a pooled material. Here, the protein content was examined on individual basis. Methods During cataract surgery, AH was sampled from patients with and without PEX, 10 eyes in each group. The proteins were digested and labelled with isotopomeric dimethyl labels, separated with high-pressure liquid chromatography and analysed in an Orbitrap mass analyzer. Results The concentration of complement factor 3, kininogen-1, antithrombin III and vitamin D-binding protein was increased in all eyes with PEX. Retinol-binding protein 3, glutathione peroxidase, calsyntenin-1 and carboxypeptidase E were decreased in eyes with PEX. Beta-crystalline B1 and CRYBB2 and gamma-crystalline D were up to eightfold upregulated in 4 of 10 in eyes with PEX. Conclusion The results indicate that oxidative stress and inflammation are contributing factors in the formation of PEX. Knowledge about the proteome in PEX is relevant for understanding this condition

Integrative "Omics"-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses

Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level ("expressome"). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions

Quantitative Mass Spectrometry Reveals Partial Translational Regulation for Dosage Compensation in Chicken

There is increasing evidence that dosage compensation is not a ubiquitous feature following sex chromosome evolution, especially not in organisms where females are the heterogametic sex, like in birds. Even when it occurs, compensation can be incomplete and limited to dosage-sensitive genes. However, previous work has mainly studied transcriptional regulation of sex-linked genes, which may not reflect expression at the protein level. Here, we used liquid chromatography–tandem mass spectrometry to detect and quantify expressed levels of more than 2,400 proteins in ten different tissues of male and female chicken embryos. For comparison, transcriptome sequencing was performed in the same individuals, five of each sex. The proteomic analysis revealed that dosage compensation was incomplete, with a mean male-to-female (M:F) expression ratio of Z-linked genes of 1.32 across tissues, similar to that at the RNA level (1.29). The mean Z chromosome-to-autosome expression ratio was close to 1 in males and lower than 1 in females, consistent with partly reduced Z chromosome expression in females. Although our results exclude a general mechanism for chromosome-wide dosage compensation at translation, 30% of all proteins encoded from Z-linked genes showed a significant change in the M:F ratio compared with the corresponding ratio at the RNA level. This resulted in a pattern where some genes showed balanced expression between sexes and some close to 2-fold higher expression in males. This suggests that proteomic analyses will be necessary to reveal a more complete picture of gene regulation and sex chromosome evolution

Иностранный язык (английский)

Данный практикум состоит из разделов, связанных между собой социальной и лингвострановедческой тематикой. Каждый раздел включает вокабуляр, ряд упражнений, способствующий развитию навыков подготовленной и неподготовленной устной речи. Дополнительные упражнения и дополнительный текстовый материал служат развитию креативного мышления на практических занятиях по английскому языку. Практикум для студентов технических специальностей дневной и заочной форм обучения

Adaptation to Photooxidative Stress: Common and Special Strategies of the Alphaproteobacteria Rhodobacter sphaeroides and Rhodobacter capsulatus .

Photosynthetic bacteria have to deal with the risk of photooxidative stress that occurs in presence of light and oxygen due to the photosensitizing activity of (bacterio-) chlorophylls. Facultative phototrophs of the genus Rhodobacter adapt the formation of photosynthetic complexes to oxygen and light conditions, but cannot completely avoid this stress if environmental conditions suddenly change. R. capsulatus has a stronger pigmentation and faster switches to phototrophic growth than R. sphaeroides. However, its photooxidative stress response has not been investigated. Here, we compare both species by transcriptomics and proteomics, revealing that proteins involved in oxidation-reduction processes, DNA, and protein damage repair play pivotal roles. These functions are likely universal to many phototrophs. Furthermore, the alternative sigma factors RpoE and RpoHII are induced in both species, even though the genetic localization of the rpoE gene, the RpoE protein itself, and probably its regulon, are different. Despite sharing the same habitats, our findings also suggest individual strategies. The crtIB-tspO operon, encoding proteins for biosynthesis of carotenoid precursors and a regulator of photosynthesis, and cbiX, encoding a putative ferrochelatase, are induced in R. capsulatus. This specific response might support adaptation by maintaining high carotenoid-to-bacteriochlorophyll ratios and preventing the accumulation of porphyrin-derived photosensitizers