Metaproteomics Methods To Discover Ecosystem Function In Aquatic Environment
Metaproteomics methods are pivotal in unraveling the intricate dynamics of ecosystem function within aquatic environments, offering a comprehensive understanding of the biochemical processes at play. By employing sophisticated techniques such as liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS), researchers can delve into the diverse microbial communities present, elucidating their roles in biogeochemical cycles and nutrient cycling. These methods facilitate the identification and quantification of proteins expressed by various organisms, shedding light on their metabolic activities, trophic interactions, and responses to environmental stimuli. Through metaproteomics, researchers can decipher the multifaceted web of interactions within aquatic ecosystems, providing invaluable insights into their resilience, stability, and responses to anthropogenic stressors, ultimately informing strategies for environmental management and conservation.
System approaches to elucidate ecosystem functioning constitute an emerging area of research within microbial ecology. Such approaches aim at investigating all levels of biological information (DNA, RNA, proteins and metabolites) to capture the functional interactions occurring in a given ecosystem and track down characteristics that could not be accessed by the study of isolated components. In this context, the study of the proteins collectively expressed by all the microorganisms present within an ecosystem (metaproteomics) is not only crucial but can also provide insights into microbial functionality. Overall, the success of metaproteomics is closely linked to metagenomics, and with the exponential increase in the availability of metagenome sequences, this field of research is starting to experience generation of an overwhelming amount of data, which requires systematic analysis. Metaproteomics has been employed in very diverse environments, and this review discusses the recent advances achieved in the context of human biology, soil, marine and freshwater environments as well as natural and bioengineered systems.
TITLE PAGE
APPROVAL PAGE
DEDICATION
ACKNOWLEDGEMENT
ABSTRACT
TABLE OF CONTENT
CHAPTER ONE
1.0 INTRODUCTION
1.1 BACKGROUND OF THE STUDY
1.2 PROBLEM STATEMENT
1.3 AIM OF THE STUDY
1.4 SIGNIFICANCE OF THE STUDY
1.5 SCOPE OF THE STUDY
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 REVIEW OF THE STUDY
2.2 AN OVERVIEW OF METAPROTEOMICS
2.3 PROTEOMIC OF MICROBIAL COMMUNITY
2.4 METAPROTEOMICS AND THE HUMAN INTESTINALMICROBIOME
2.5 METAPROTEOMICS IN ENVIRONMENTAL MICROBIOME STUDIES
CHAPTER THREE
3.0 METHODS
3.1 PROTEIN EXPRESSION IN THE HUMAN MICROBIOME
3.2 PROTEIN EXPRESSION IN SOIL
3.3 PROTEIN EXPRESSION IN MARINE AND FRESHWATER
CHAPTER FOUR
4.0 RESULT
CHAPTER FIVE
CONCLUSIONS
RECOMMENDATION
1.0 INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Microorganisms occupy virtually every habitat on our planet, and their activities largely determine the environmental conditions of today’s world. Indeed, microorganisms are heavily involved in biogeochemistry, ensuring the recycling of elements such as carbon and nitrogen (Madsen, 2011). In addition, microorganisms are extensively used to degrade anthropogenic waste prior to release into the environment (Hussain et al., 2010). In their natural habitat, microorganisms coexist in mixed communities, the complexity of which is specific to each environment, for example from six estimated individual taxa for an acid mine drainage biofilm (Ram et al., 2005), up to 106 estimated taxa per gram of soil. As most of the microorganisms present in the environment have not been cultured, their investigation requires the use of molecular techniques that bypass the traditional isolation and cultivation of individual species (Amann et al., 1995). Moreover, even when isolation is possible, a single species removed from its natural environment might not necessarily display the same characteristics under laboratory conditions as it does within its ecological niche. Therefore, the study of mixed microbial communities within their natural environment is key to the investigation of the diverse roles played by microorganisms, and to the identification of the microbial potential for biotechnological application, including but not limited to: pharmaceutical, diagnostics, waste treatment, bioremediation and renewable energy generation. An emerging field of research in microbial ecology encompasses system approaches, whereby all levels of biological information are investigated (DNA, RNA, proteins and metabolites) to capture the functional interactions occurring in a given ecosystem and identify characteristics that could not be accessed by the study of isolated components (Röling et al., 2010). Recent technological advances, including the development of high-throughput ‘omics’ methods, make such system approaches possible, where mixed microbial communities are viewed as one meta-organism. Metaproteomics are employed to determine respectively the DNA sequences of the meta-organism under study, the collectively transcribed RNA, the translated proteins and the metabolites resulting from cellular processes. All of the generated data can then be used to identify the metabolic pathways and cellular processes at work within an ecosystem.
1.2 Problem statement
Aquatic ecosystems support a substantial source of the earth’s biological diversity. They are an essential reservoir and share an enormous proportion of earth’s biological productivity. Both aquatic resources and its biodiversity are interrelated to each other and they perform a myriad of functions and are valuable and essential for the sustainability of biotic communities. Aquatic biodiversity in both freshwater and marine environments are under continuous decline because of overexploitation of species, introduced exotic plant or animal, pollution sources from cities, industries and agricultural zones, loss and changes in ecological niche. Their conservation and management in the form of bio reserve points and bioregional management and worldwide monitoring are needed for the protection of the aquatic biodiversity. This study is presenting information on biodiversity in aquatic habitats and their resources, in marine and fresh water ecosystems, their importance conservation and restoration mechanisms using metaproteomics methods.
1.3 Aim of the study
The main aim of the study is to develop and apply metaproteomic platforms to better understand environmental systems and their robustness to change.
1.4 Scope of the study
The scope of this study covers studying the quantity of the molecular cellular components (e.g. DNA, mRNA, proteins and metabolites) in environmental samples can reveal significant information on ecosystem function.
1.5 Significance of the study
This study will provide a means we can gain a much more comprehensive understanding of environmental responses to processes such as climate change or pollution etc. The field known as Environmental Omics is mostly dominated by DNA sequencing. However, proteins are the functional entities in cells and therefore identifying and quantifying proteins gives a much more accurate insight into how ecosystems respond to environmental perturbations. Gaining a snapshot of ecosystem function through measuring the proteins in an environmental sample is referred to as metaproteomics.
The project would suit ideally a biosciences/chemistry graduate with a strong interest in novel and multidisciplinary approaches to environmental engineering, analytics, or mapping ecological responses using new, cutting edge technologies.
This study will serve as a training in quantitative analytical techniques e.g. high performance liquid, mass spectrometry. This includes experimental design and analysis of large amounts of data with bioinformatics pipelines. They will become experts in handling proteins and interpreting complex data. Metaproteomics is a tool that can be transferred to many different fields so flexibility within the project is high.
Metaproteomics is just one of many omics tools which are gaining momentum in their application in the laboratory and the field. The candidate will be developing skills that can be applied for research and development in many different field including the use of cutting edge analytical equipment.
Metaproteomics is a powerful approach for studying microbial communities in aquatic environments and understanding their ecosystem functions. It involves the analysis of the collective proteins (the metaproteome) expressed by the entire microbial community within a given sample. Here are some methods and steps involved in using metaproteomics to discover ecosystem functions in aquatic environments:
- Sample Collection:
- Collect water samples from the aquatic environment you want to study. These samples should represent different locations, depths, or conditions within the ecosystem to capture the diversity of the microbial community.
- Sample Preparation:
- Filter the collected water samples to concentrate microbial cells. This can be done using various filter pore sizes to capture different microbial size fractions.
- Lyse the microbial cells to release their proteins. Different lysis methods can be used, such as chemical, mechanical, or enzymatic methods.
- Protein Extraction:
- Extract proteins from the lysed microbial cells. Different extraction methods can be used depending on the sample type and the organisms present.
- Protein Separation:
- Separate the extracted proteins using gel electrophoresis or liquid chromatography. This step helps in fractionating the complex protein mixture.
- Protein Digestion:
- Proteins are then enzymatically digested into peptides, typically using trypsin. This step breaks down proteins into smaller, more manageable units for mass spectrometry analysis.
- Mass Spectrometry (MS) Analysis:
- Analyze the peptides using mass spectrometry. This step identifies and quantifies the peptides based on their mass and charge. High-resolution mass spectrometers are commonly used for metaproteomics.
- Database Search:
- Compare the mass spectrometry data against a database of known microbial proteins. This helps in identifying the microorganisms present in the sample and their functional proteins.
- Functional Annotation:
- Assign functions to the identified proteins based on their known or predicted functions. This step involves using bioinformatics tools and databases, such as KEGG, COG, or SEED, to link the proteins to metabolic pathways and functions.
- Quantitative Analysis:
- Use quantitative mass spectrometry techniques (e.g., label-free quantification or isobaric labeling) to determine the abundance of identified proteins in the samples. This helps in understanding the relative contributions of different microorganisms and their functions in the ecosystem.
- Data Integration:
- Combine metaproteomic data with other environmental data, such as metagenomic data, physicochemical measurements, and environmental metadata, to gain a comprehensive view of ecosystem functions.
- Statistical Analysis:
- Perform statistical analyses to identify significant differences in protein abundance between samples or conditions, which can reveal shifts in ecosystem function.
- Interpretation:
- Interpret the results to understand the key microbial players and their functional roles within the aquatic ecosystem. Look for patterns, correlations, and associations between proteins and environmental parameters.
- Validation:
- Validate findings through targeted experiments, such as enzyme assays or gene expression analysis, to confirm the functional roles of specific proteins or pathways.
- Data Visualization:
- Present the results through visualizations like heatmaps, network diagrams, or ecological models to communicate the ecosystem’s functional dynamics effectively.
Metaproteomics is a complex and data-intensive field, but it provides valuable insights into the functional capabilities of microbial communities in aquatic environments, helping researchers understand the roles of microorganisms in ecosystem processes such as nutrient cycling, carbon turnover, and pollutant degradation.