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Proteomic and genomic techniques towards a better life in environmental research

Hausarbeit 2019 23 Seiten

Biologie - Genetik / Gentechnologie





2.1 Proteomics techniques

2.2 Genomics Techniques




The dynamic role of molecules to support the life is documented since the initial stages of biological research. To demonstrate the importance of these molecules, Berzelius in 1838 given the title “protein”, which is originated from the Greek word, proteios, meaning “the first rank” (Cristea et al., 2004) Proteome is the entire set of proteins encoded by the genome and proteomics is the discipline which studies the global set of proteins, their expression, function and structure. Knowledge of proteins is thus crucial in understanding the mechanism of any biological process. Although advances in genome sequencing have allowed the identification of a number of open reading frames (ORFs), but this information is far from complete. On an average, about 40% of the gene sequences detected in the genomic databases code for proteins of hypothetical or unknown function. Further, the number of genes present in a genome is less than the array of proteins found in the cell (Anderson et al., 2016). Besides compositional complexity and concentration range, protein dynamics, i.e. the protein expression changes over time, add to the complexity of a proteome (Agrawal et al., 2012). Bacterial genomes code for about 600–6,000 genes but only a part of the genome, usually 50–80% are expressed under specific life circumstances depending on the environmental stimuli that reach the cell (Anderson et al., 2016). The low complexity makes bacteria a reasonable model system to address crucial and elementary issues of life processes by using proteomics approaches (Hecker et al., 2008). However, proteins act as aggregates in cellular machineries, they are targeted to their final destinations inside or outside the cell and they can be reversibly or even irreversibly modified, damaged, repaired and in hopeless cases even degraded (Hecker et al., 2008).

Proteomics is crucial for early disease diagnosis, prognosis and to monitor the disease development. Furthermore, it also has a vital role in drug development as target molecules. Proteomics is the characterization of proteome, including expression, structure, functions, interactions and modifications of proteins at any stage. (Domon and Aebersold, 2006). The proteome also fluctuates from time to time, cell to cell and in response to external stimuli. Proteomics in eukaryotic cells is complex due to post-translational modifications, which arise at different sites by numerous ways (baak et al., 2003).

Proteomics is one of the most significant methodologies to comprehend the gene function although, it is much more complex compared with genomic (Ethier et al., 2006). Fluctuations in gene expression level can be determined by analysis of transcriptome or proteome to discriminate between two biological states of the cell. Microarray chips have been developed for large-scale analysis of whole transcriptome. However, increase synthesis of mRNA cannot measure directly by microarray (Devoe and Lee, 2006). Proteins are effectors of biological function and their levels are not only dependent on corresponding mRNA levels but also on host translational control and regulation. Thus, the proteomics would be considered as the most relevant data set to characterize a biological system (Freire and Wheeler, 2006).

The term genomics was coined by Tom Roderick, a geneticist at the Jackson Laboratory (Yadav, 2007). According to National Human Genome Research Institute 2010 defines Genomics as an interdisciplinary field of science focusing on the structure, function, evolution, mapping, and editing of genomes the collective characterization and quantification of genes, which direct the production of proteins with the assistance of enzymes and messenger molecules. In turn, proteins make up body structures such as organs and tissues as well as control chemical reactions and carry signals between cells. Genomics also involves the sequencing and analysis of genomes through uses of high throughput DNA sequencing and bioinformatics to assemble and analyze the function and structure of entire genomes (Goldenfeld and Woese, 2007). A genome is an organism's complete set of DNA, including all of its genes.

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Figure. 1: The hierarchy of ‘‘Omics approaches’’

Source: Green and Keller, 2006

Environmental genomics and genome-wide expression approaches deal with large-scale sequence-based information obtained from environmental samples, at organismal, population or community levels. To date, environmental genomics, transcriptomics and proteomics are arguably the most powerful approaches to discover completely novel ecological functions and to link organism capabilities, organism–environment interactions, functional diversity, ecosystem processes, evolution and Earth history. Thus, environmental genomics is not merely a toolbox of new technologies but also a source of novel ecological concepts and hypotheses. By removing previous dichotomies between ecophysiology, population ecology, community ecology and ecosystem functioning, environmental genomics enables the integration of sequence-based information into higher ecological and evolutionary levels (Goldenfeld and Woese, 2007). However, environmental genomics, along with transcriptomics and proteomics, must involve pluridisciplinary research, such as new developments in bioinformatics, in order to integrate high throughput molecular biology techniques into ecology the validity of environmental genomics and post-genomics for studying ecosystem functioning is discussed in terms of major advances and expectations. Novel avenues for improving the use of these approaches to test theory-driven ecological hypotheses are also explored (Horvatovich et al., 2007).

The application of genomics and derivative technologies yields insight into ecosystems. The use of genomics, functional genomics, proteomic and systems modeling approaches allows for the analysis of community population structure, functional capabilities and dynamics. The process typically begins with sequencing of DNA extracted from an environmental sample, either after cloning the DNA into a library or by affixing to beads and direct sequencing. After the sequence is assembled, the computational identification of marker genes allows for the identification and phylogenetic classification of the members of the community and enables the design of probes for subsequent population structure experiments. The assignment of sequence fragments into groups that correspond to a single type of organism (a process called ‘binning’) is facilitated by identification of marker genes within the fragments, as well as by other characteristics such as G+C content bias and codon usage preferences. Computational genome annotation, consisting of the prediction of genes and assignment of function using characterized homologs and genomic context, allows for the description of the functional capabilities of the community. Knowledge of the genes present also enables functional genomics and proteomic techniques, applied to extracts of protein and RNA transcripts from the sample (Fredrickson and Romine, 2005).

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Figure 2: Application of genomics and derivative technologies.

Source: ( Fredrickson and Romine, 2005)


2.1 Proteomics techniques

2D Electrophoresis: Early environmental proteomics research focused almost exclusively on the use of 2D-GE–MS technology. In this case, environmental samples were collected and evaluated using a 2D gel display followed by downstream MS analysis. The main focus for most of the described papers was not to extensively identify each and every protein spot in the complete gels but rather to focus on interesting spots that changed location or intensity as a function of sample or growth condition (Wilmes and Bond, 2004).

Wilmes and Bond (2006) also used 2D-PAGE coupled with MALDITOF MS on a laboratory-scale activated sludge system optimized for EBPR. This method enabled the successful extraction and purification of the entire proteome, its separation by 2D-PAGE, and the mapping of this metaproteome. Highly expressed protein spots were excised and identified using quadrupole time-of-flight MS with de novo peptide sequencing. The isolated proteins were putatively identified as an outer membrane protein (porin), an acetyl coenzyme A acetyltransferase, and a protein component of an ABC-type branchedchain amino acid transport system. These proteins were postulated to stem from the dominant and uncultured Rhodocyclus - type polyphosphate-accumulating organism in the activated sludge.

Markert et al., performed a 2D gel-based proteomics approach on the bacterial endosymbiont of the deep-sea tube worm Riftia pachyptila, revealing that three major sulfide oxidation proteins constitute about 12% of the total cytosolic proteome.

Klaassens et al., (2007) showed for the first time the extraction of proteins, reproducible 2D gel electrophoresis, and tentative identification using MALDI-TOF MS of the metaproteome of a complex intestinal ecosystem of an uncultured infant fecal microbiota.

Multidimensional Liquid Chromatography–Mass Spectrometry -: The revolution in the use of gel-less online multidimensional LC-MS/MS technology for studying microbial isolates opened up a new regimen of comprehensive proteome characterization (Cravatt et al., 2007), now enabling the identification of a few thousand proteins from an individual cultivated microorganism (DeLong, 2004). This permits a detailed, fairly deep glimpse into the molecular activities of the bacteria and now provides a robust technology which can be extended to environmental samples.



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proteomic Genomics



Titel: Proteomic and genomic techniques towards a better life in environmental research