Authors: Dharmendra kumar
ICAR-IARI, Division of Microbiology, New Delhi-12
Metaproteomics (also Community Proteomics, Environmental Proteomics, or Community Proteogenomics) is the study of all protein samples recovered directly from environmental sources. Metaproteomics is used to classify experiments that deal with all the genes and proteins identified from complex communities, where individuals cannot be binned into species or organisms types.The metaproteomics approach is comparable to gene-centric environmental genomics , or metagenomics .
Proteomics of microbial community
The first proteomics experiment was conducted with the invention of two-dimensional polyacrylamid gel electrophoresis (2D-PAGE).The 1980s and 1990s saw the development of mass spectrometry and mass spectrometry based proteomics. The current proteomics of microbial community makes use of both gel-based (one-dimensional and two-dimensional) and non-gel liquid chromatography based separation, where both rely on mass spectrometry based peptide identification.While proteomics is largely a discovery-based approach that is followed by other molecular or analytical techniques to provide a full picture of the subject system, it is not limited to simple cataloging of proteins present in a sample. With the combined capabilities of "top-down" and "bottom-up" approaches, proteomics can pursue inquiries ranging from quantitation of protein expression between growth conditions (whether nutritional, spatial, temporal, or chemical) to protein structural information.
Not only does the translation from mRNA cause differences, but many proteins are also subjected to a wide variety of chemical modifications after translation. Many of these post-translational modifications are critical to the protein's function. Phosphorylation.
UbiquitinationUbiquitin is a small protein that can be affixed to certain protein substrates by enzymes called E3 ubiquitin ligases Determining which proteins are poly-ubiquitinated helps understand how protein pathways are regulated. This is, therefore, an additional legitimate "proteomic" study. Similarly, once a researcher determines which substrates are ubiquitinated by each ligase, determining the set of ligases expressed in a particular cell type is helpful.
Additional modificationsIn addition to phosphorylation and ubiquitination proteins can be subjected to methylation, acetylation,glycosylation,oxidation and nitrosylation. Some proteins undergo all these modifications, often in time-dependent combinations. This illustrates the potential complexity of studying protein structure and function.
Distinct proteins are made under distinct settings
Even studying a particular cell type, that cell may make different sets of proteins at different times, or under different conditions. Furthermore, as mentioned, any one protein can undergo a wide range of post-translational modifications.
Limitations of genomics and proteomics studies
Proteomics gives a different level of understanding than genomics for many reasons: The level of transcription of a gene gives only a rough estimate of its level of translation into a protein. An mRNA produced in abundance may be degraded rapidly or translated inefficiently, resulting in a small amount of protein.
One of the earliest method for protein analysis has been Edman degradation (introduced in 1967) where a single peptide is subjected to multiple steps of chemical degradation to resolve its sequence. These methods have mostly been supplanted by technologies that offer higher throughput.More recent methods use mass spectrometry based techniques, a development that was made possible by the discovery of "soft ionization" methods such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) developed in the 1980s. These methods gave rise to the top-down and the bottom-up proteomics workflows where often additional separation is performed before analysis (see below).Separation methods- For the analysis of complex biological samples, a reduction of sample complexity is required. This can be performed off-line by one-dimensional or two dimensional separation. More recently, on-line methods have been developed where individual peptides (in bottom-up proteomics approaches) are separated using Reversed-phase chromatography and then directly ionized using ESI .the direct coupling of separation and analysis explains the term "on-line" analysis.
There are several hybrid technologies that use antibody-based purification of individual analytes and then perform mass spectrometric analysis for identification and quantification. Examples of these methods are the MSIA (mass spectrometric immunoassay) developed by Randall Nelson in 1995 and the SISCAPA (Stable Isotope Standard Capture with Anti-Peptide Antibodies) method, introduced by Leigh Anderson in 2004.
The National Institutes of Health has defined a biomarker as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.”Understanding the proteome, the structure and function of each protein and the complexities of protein–protein interactions is critical for developing the most effective diagnostic techniques and disease treatments in the future.
In what is now commonly referred to as proteogenomics proteomic technologies such as mass spectrometry are used for improving gene annotations. Parallel analysis of the genome and the proteome facilitates discovery of post-translational modifications and proteolytic events,especially when comparing multiple species (comparative proteogenomics).
Structural proteomics includes the analysis of protein structures at large-scale. It compares protein structures and helps identify functions of newly discovered genes. The structural analysis also helps to understand that where drugs bind to proteins and also show where proteins interact with each other. This understanding is achieved using different technologies such as X-ray crystallography and NMR spectroscopy.
Expression proteomics includes the analysis of protein expression at larger scale. It helps identify main proteins in a particular sample, and those proteins differentially expressed in related samples—such as diseased vs. healthy tissue. If a protein is found only in a diseased sample then it can be a useful drug target or diagnostic marker. Proteins with same or similar expression profiles may also be functionally related. There are technologies such as 2D-PAGE and mass spectrometry that are used in expression proteomics.
Interaction proteomics is the analysis of protein interactions at larger scale. The characterization of protein-protein interactions are useful to determine the protein functions and it also explains the way proteins assemble in bigger complexes. Technologies such as affinity purification, mass spectrometry, and the yeast two-hybrid system are particularly useful in interaction proteomics.
Current proteomic technologies
Proteomics has steadily gained momentum over the past decade with the evolution of several approaches. Few of these are new and others build on traditional methods. Mass spectrometry-based methods and micro arrays are the most common technologies for large-scale study of proteins.
Mass spectrometry and protein profilingMass spectrometry
There are two mass spectrometry-based methods currently used for protein profiling. The more established and widespread method uses high resolution, two-dimensional electrophoresis to separate proteins from different samples in parallel, followed by selection and staining of differentially expressed proteins to be identified by mass spectrometry. Despite the advances in 2DE and its maturity, it has its limits as well. The central concern is the inability to resolve all the proteins within a sample, given their dramatic range in expression level and differing properties.
Protein chipsBalancing the use of mass spectrometers in proteomics and in medicine is the use of protein micro arrays. The aim behind protein micro arrays is to print thousands of protein detecting features for the interrogation of biological samples. Antibody arrays are an example in which a host of different antibodies are arrayed to detect their respective antigens from a sample of human blood. Another approach is the arraying of multiple protein types for the study of properties like protein-DNA, protein-protein and protein-ligand interactions. Ideally, the functional proteomic arrays would contain the entire complement of the proteins of a given organism.
Overall, the field of metaproteomics is gaining momentum at an exponential rate within very diverse environments. To date, the application of complex
system approaches is still scarce and requires a coordinated experimental design that brings together expertise from each of the many technologies involved. However, it should be kept in mind that an exhaustive investigation of the entire metaproteome is unlikely due to the unfeasibility of developing a universal protein analysis protocol. Furthermore, it must be considered that a metaproteomics may include intracellular, extracellular and membrane-bound proteins, and ideally, the three protein fractions should be analysed for each sample. When possible, opting for gel-free protein fractionation seems to lead to a higher level of protein identification when compared with gel-based methods. For example, when analysing the metaproteome of activated sludge, the use of 2-DGE resulted in the identification of 38 proteins while the 2D-nano-LC method led to the identification of 5029 proten.
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