Evolutionary analysis of enzymes using Chisel

MOTIVATION: Availability of large volumes of genomic and enzymatic data for taxonomically and phenotypically diverse organisms allows for exploration of the adaptive mechanisms that led to diversification of enzymatic functions. We present Chisel, a computational framework and a pipeline for an automated, high-resolution analysis of evolutionary variations of enzymes. Chisel allows automatic as well as interactive identification, and characterization of enzymatic sequences. Such knowledge can be utilized for comparative genomics, microbial diagnostics, metabolic engineering, drug design and analysis of metagenomes. RESULTS: Chisel is a comprehensive resource that contains 8575 clusters and subsequent computational models specific for 939 distinct enzymatic functions and, when data is sufficient, their taxonomic variations. Application of Chisel to identification of enzymatic sequences in newly sequenced genomes, analysis of organism-specific metabolic networks, 'binning' of metagenomes and other biological problems are presented. We also provide a thorough analysis of Chisel performance with other similar resources and manual annotations on Shewanella oneidensis MR1 genome.     

Validation of 4 type-STR analysis for identification of 50 Korean

Short tandem repeat (STR) analysis provides genetic fingerprinting of individuals and is an indispensable technique for forensic human identification. Recently, this technique has been used in social areas, such as the identification of The Korean War, descendants of national merit, and missing children. STR analysis is performed by analyzing iteration number of repeating bases in the human genome, and currently FBI provides the Combined DNA Index System (CODIS) based on DNA databases. Among them, we used the autosomal short tandem repeats of loci D13S317, D16S539, D21S11, and amelogenin to validate this technique for identification. The samples were collected from unrelated 50 Korean individuals, and 4 STR loci of these samples were analyzed by ABI 3130 genetic analyzer. We demonstrated that 47 samples out of 50 were classified completely with only 4 STR markers, and perfect sex identification could be accomplished with amelogenin analysis.     

Algorithms and tools for analysis and management of mass spectrometry data

Mass spectrometry (MS) is a technique that is used for biological studies. It consists in associating a spectrum to a biological sample. A spectrum consists of couples of values (intensity, m/z), where intensity measures the abundance of biomolecules (as proteins) with a mass-to-charge ratio (m/z) present in the originating sample. In proteomics experiments, MS spectra are used to identify pattern expressions in clinical samples that may be responsible of diseases. Recently, to improve the identification of peptides/proteins related to patterns, MS/MS process is used, consisting in performing cascade of mass spectrometric analysis on selected peaks. Latter technique has been demonstrated to improve the identification and quantification of proteins/peptide in samples. Nevertheless, MS analysis deals with a huge amount of data, often affected by noises, thus requiring automatic data management systems. Tools have been developed and most of the time furnished with the instruments allowing: (i) spectra analysis and visualization, (ii) pattern recognition, (iii) protein databases querying, (iv) peptides/proteins quantification and identification. Currently most of the tools supporting such phases need to be optimized to improve the protein (and their functionalities) identification processes. In this article we survey on applications supporting spectrometrists and biologists in obtaining information from biological samples, analyzing available software for different phases. We consider different mass spectrometry techniques, and thus different requirements. We focus on tools for (i) data preprocessing, allowing to prepare results obtained from spectrometers to be analyzed; (ii) spectra analysis, representation and mining, aimed to identify common and/or hidden patterns in spectra sets or in classifying data; (iii) databases querying to identify peptides; and (iv) improving and boosting the identification and quantification of selected peaks. We trace some open problems and report on requirements that represent new challenges for bioinformatics.     

Correct identification of genes from serial analysis of gene expression tag sequences

SAGE (serial analysis of gene expression) is a remarkable technique for genome-wide analysis of gene expression. It is crucial to understand the extent to which SAGE can accurately indicate a gene or expressed sequence tag (EST) with a single tag. We analyzed the effect of the size of SAGE tag on gene identification. Our observation indicates that SAGE tags are in general not long enough to achieve the degree of uniqueness of identification originally envisaged. Our observations also indicate that the limitation of using SAGE tag to identify a gene can be overcome by converting SAGE tags into longer 3' EST sequences with the generation of longer cDNA fragments from SAGE tages for gene identification (GLGI) method.     

CD8 T cell immunome analysis of Listeria monocytogenes

The identification of T cell epitopes is crucial for the understanding of the host response during infections with pathogenic microorganisms. Generally, the identification of relevant T cell responses is based on the analysis of T cell lines propagated in vitro. We used an ex vivo approach for the analysis of the CD8 T cell response against Listeria monocytogenes that is based upon the fractionation of naturally processed antigenic peptides and subsequent analysis with T cells in an enzyme-linked immunospot (ELISPOT) assay. Our data indicate that the direct ex vivo ELISPOT analysis of peptides extracted from infected tissues represents a versatile and potent test system for the analysis of the CD8 T cell immunome of microorganisms that furthermore requires neither the knowledge of the microbial genome nor of the specificity of responding T cells.     

Comprehensive proteomic analysis of Penicillium verrucosum

Mass spectrometric identification of proteins in species lacking validated sequence information is a major problem in veterinary science. In the present study, we used ochratoxin A producing Penicillium verrucosum to identify and quantitatively analyze proteins of an organism with yet no protein information available. The work presented here aimed to provide a comprehensive protein identification of P. verrucosum using shotgun proteomics. We were able to identify 3631 proteins in an “ab initio” translated database from DNA sequences of P. verrucosum. Additionally, a sequential window acquisition of all theoretical fragment‐ion spectra analysis was done to find differentially regulated proteins at two different time points of the growth curve. We compared the proteins at the beginning (day 3) and at the end of the log phase (day 12).     

rpoB sequence analysis as a novel basis for bacterial identification

Comparison of the sequences of conserved genes, most commonly those encoding 16S rRNA, is used for bacterial genotypic identification. Among some taxa, such as the Enterobacteriaceae, variation within this gene does not allow confident species identification. We investigated the usefulness of RNA polymerase beta-subunit encoding gene ( rpoB  ) sequences as an alternative tool for universal bacterial genotypic identification. We generated a database of partial rpoB for 14 Enterobacteriaceae species and then assessed the intra- and interspecies divergence between the rpoB and the 16S rRNA genes by pairwise comparisons. We found that levels of divergence between the rpoB sequences of different strains were markedly higher than those between their 16S rRNA genes. This higher discriminatory power was further confirmed by assigning 20 blindly selected clinical isolates to the correct enteric species on the basis of rpoB sequence comparison. Comparison of rpoB sequences from Enterobacteriaceae was also used as the basis for their phylogenetic analysis and demonstrated the genus Klebsiella to be polyphyletic. The trees obtained with rpoB were more compatible with the currently accepted classification of Enterobacteriaceae than those obtained with 16S rRNA. These data indicate that rpoB is a powerful identification tool, which may be useful for universal bacterial identification.     

A method for the comprehensive proteomic analysis of membrane proteins

We describe a method that allows for the concurrent proteomic analysis of both membrane and soluble proteins from complex membrane-containing samples. When coupled with multidimensional protein identification technology (MudPIT), this method results in (i) the identification of soluble and membrane proteins, (ii) the identification of post-translational modification sites on soluble and membrane proteins, and (iii) the characterization of membrane protein topology and relative localization of soluble proteins. Overlapping peptides produced from digestion with the robust nonspecific protease proteinase K facilitates the identification of covalent modifications (phosphorylation and methylation). High-pH treatment disrupts sealed membrane compartments without solubilizing or denaturing the lipid bilayer to allow mapping of the soluble domains of integral membrane proteins. Furthermore, coupling protease protection strategies to this method permits characterization of the relative sidedness of the hydrophilic domains of membrane proteins.     

Structural analysis of multiprotein complexes by cross-linking, mass spectrometry, and database searching

Most protein complexes are inaccessible to high resolution structural analysis. We report the results of a combined approach of cross-linking, mass spectrometry, and bioinformatics to two human complexes containing large coiled-coil segments, the NDEL1 homodimer and the NDC80 heterotetramer. An important limitation of the cross-linking approach, so far, was the identification of cross-linked peptides from fragmentation spectra. Our novel approach overcomes the data analysis bottleneck of cross-linking and mass spectrometry. We constructed a purpose-built database to match spectra with cross-linked peptides, define a score that expresses the quality of our identification, and estimate false positive rates. We show that our analysis sheds light on critical structural parameters such as the directionality of the homodimeric coiled coil of NDEL1, the register of the heterodimeric coiled coils of the NDC80 complex, and the organization of a tetramerization region in the NDC80 complex. Our approach is especially useful to address complexes that are difficult in addressing by standard structural methods.     

Isoform analysis of LC-MS/MS data from multidimensional fractionation of the serum proteome

We developed a visualization approach for the identification of protein isoforms, precursor/mature protein combinations, and fragments from LC-MS/MS analysis of multidimensional fractionation of serum and plasma proteins. We also describe a pattern recognition algorithm to automatically detect and flag potentially heterogeneous species of proteins in proteomic experiments that involve extensive fractionation and result in a large number of identified serum or plasma proteins in an experiment. Examples are given of proteins with known isoforms that validate our approach and present a subset of precursor/mature protein pairs that were detected with this approach. Potential applications include identification of differentially expressed isoforms in disease states.     

Molecular analysis in the conservation of sturgeons and paddlefish

Sturgeon and paddlefish populations worldwide have declined because of anthropogenic influences. The structure and magnitude of genetic diversity of natural populations serves to buffer these fishes against environmental variation and should be maintained. Modern molecular biological techniques provide the ability to sensitively characterize and quantify the extent of genetic variation in natural populations. We provide a summary of those problems in sturgeon population biology that are amenable to investigation with DNA approaches, and their applications to date. These have included genetic identification and discrimination of taxa, identification of hybrids, stock identification, mixed-stock analysis, and estimation of gene flow and homing fidelity. To date, almost all studies have been restricted to North American fauna. Improvements to these technologies, including nondestructive sampling, should permit more widespread application of molecular approaches to problems of acipenseriform conservation. We suggest that the use of more sensitive molecular tools such as analyses of hypervariable repetitive and non-coding single copy nuclear DNA may assist management even in those taxa which exhibit overall low levels of genetic diversity.     

Identification of Myosotis seeds by means of digital image analysis

Despite their name, the identification of seeds of Myosotis species (forget-me-not) has hitherto received little attention from archaeobotanists. In an attempt to assemble a collection of reliable identification criteria, digital image analysis was applied to photographs of Myosotis seeds by means of Fovea Pro 4.0. This program computes 23 features that describe the size and shape of the seeds shown in scale-normalized photographs. We computed the features for 1,453 individual seeds, and performed statistical analyses of the resulting data set with Discriminant Analysis, Correspondence Analysis, and t-Distributed Stochastic Neighbour Embedding (t-SNE). The combination of analyses provides clues as to how most of the seven western European species of Myosotis can successfully be distinguished. Using these clues, an identification key was developed for the identification of waterlogged Myosotis seeds.     

Genome-scale analysis of demographic history and adaptive selection

One of the main topics in population genetics is identification of adaptive selection among populations. For this purpose, population history should be correctly inferred to evaluate the effect of random drift and exclude it in selection identification. With the rapid progress in genomics in the past decade, vast genomescale variations are available for population genetic analysis, which however requires more sophisticated models to infer species’ demographic history and robust methods to detect local adaptation. Here we aim to review what have been achieved in the fields of demographic modeling and selection detection. We summarize their rationales, implementations, and some classical applications. We also propose that some widely-used methods can be improved in both theoretical and practical aspects in near future.     

Proteomic analysis of a fraction enriched in cell envelope proteins of Acinetobacter baumannii

Acinetobacter baumannii is a multiresistant opportunistic nosocomial pathogen. A protein fraction was purified and analyzed by 2-DE. Twenty-nine major protein spots were selected for protein identification using trypsin digestion and MS analysis. As the A. baumannii genome has not yet been described, protein identification was performed by homology with other Acinetobacter species in the NCBi database. We identified ribosomal proteins, chaperones, elongation factors and outer membrane proteins (Omp), such as OmpA and the 33-36-kDa OMP. Proteomic analysis of A. baumannii provides a platform for further studies in antimicrobial resistance.     

Multicolour digital image analysis system for identification of bacteria and concurrent assessment of their respiratory activity

AIMS: To develop a rapid and simple multicolour digital image analysis system for simultaneous identification of bacteria and assessment of their metabolic activity. METHODS AND RESULTS: We developed an image analyser capable of distinguishing triple-stained bacterial cells. Bacteria were stained with a nucleic acid stain, a fluorescent antibody and a fluorescent metabolic indicator for enumeration, species identification and assessment of metabolic activity. This multicolour image analyser was used to simultaneously identify Escherichia coli O157:H7 in milk samples and assess their respiratory activity. The images of the triple-stained bacteria were captured using a combination of blue light and u.v. excitation and an epifluorescence microscope and were processed by our image analyser. We found a good correlation between the counts of actively respiring (r = 0.93) and total (r = 0.94) E. coli O157:H7 measured by digital image analysis and visual observation. CONCLUSION: The multicolour digital image analysis system described here was able to quantify active pathogenic micro-organisms within 2 h. SIGNIFICANCE AND IMPACT OF THE STUDY: This multicolour image analysis allows the rapid and simultaneous quantification of bacteria, identification of species and assessment of metabolic activity.     

Microarray analytic system for multiplex analysis by real-time polymerase chain reaction with reagents immobilized in microreactors

A microarray analytic system that uses a silicon chip with immobilized in microreactor test-system for multiplex analysis of DNA by real-time polymerase chain reaction (RT-PCR) was developed and optimized. We suggested the method of immobilization of PCR-components of a test-system, chose the stabilizer, and conducted the optimization of the composition of reaction mixture to achieve permanent stability of a microarray. We conducted optimization of preparation of samples using magnetic sorbent and indicated that, with 2.6 x 10(4) copies/ml, 60 min are necessary to obtain positive identification including time for preparation of model probes. The abilities of the created system were demonstrated on the example of microarray analysis of samples with different content of DNA, low absolute limits of identification (20 DNA copies in microreactor), and high reproducibility of the analysis.     

Enhanced identification of peptides lacking basic residues by LC-ESI-MS/MS analysis of singly charged peptides

Peptide sequences lacking basic residues (arginine, lysine, or histidine, referred to as "base-less") are of particular importance in proteomic experiments targeting protein C-termini or employing nontryptic proteases such as GluC or chymotrypsin. We demonstrate enhanced identification of base-less peptides by focused analysis of singly charged precursors in liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Singly charged precursors are often excluded from fragmentation and sequence analysis in LC-MS/MS. We generated different pools of base-less and base-containing peptides by tryptic and nontryptic digestion of bacterial proteomes. Focused LC-MS/MS analysis of singly charged precursor ions yielded predominantly base-less peptide identifications. Similar numbers of base-less peptides were identified by LC-MS/M Sanalysis targeting multiply charged precursors. There was little redundancy between the base-less sequences derived by both MS/MS schemes. In the present experimental outcome, additional LC-MS/MS analysis of singly charged precursors substantially increased the identification rate of base-less sequences derived from multiply charged precursors. In conclusion, LC-MS/MS based identification of base-less peptides is substantially enhanced by additional focused analysis of singly charged precursors.     

High throughput peptide mass fingerprinting and protein macroarray analysis using chemical printing strategies

We describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery.     

Refined identification of Vibrio bacterial flora from Acanthasther planci based on biochemical profiling and analysis of housekeeping genes

We used a polyphasic approach for precise identification of bacterial flora (Vibrionaceae) isolated from crown-of-thorns starfish (COTS) from Lizard Island (Great Barrier Reef, Australia) and Guam (U.S.A., Western Pacific Ocean). Previous 16S rRNA gene phylogenetic analysis was useful to allocate and identify isolates within the Photobacterium, Splendidus and Harveyi clades but failed in the identification of Vibrio harveyi-like isolates. Species of the V harveyi group have almost indistinguishable phenotypes and genotypes, and thus, identification by standard biochemical tests and 16S rRNA gene analysis is commonly inaccurate. Biochemical profiling and sequence analysis of additional topA and mreB housekeeping genes were carried out for definitive identification of 19 bacterial isolates recovered from sick and wild COTS. For 8 isolates, biochemical profiles and topA and mreB gene sequence alignments with the closest relatives (GenBank) confirmed previous 16S rRNA-based identification: V. fortis and Photobacterium eurosenbergii species (from wild COTS), and V natriegens (from diseased COTS). Further phylogenetic analysis based on topA and mreB concatenated sequences served to identify the remaining 11 V harveyi-like isolates: V. owensii and V. rotiferianus (from wild COTS), and V. owensii, V. rotiferianus, and V. harveyi (from diseased COTS). This study further confirms the reliability of topA-mreB gene sequence analysis for identification of these close species, and it reveals a wider distribution range of the potentially pathogenic V. harveyi group.