Proteomics. Importance for the Future of Genetics Research


Academic Paper, 2020
19 Pages, Grade: 14.0

Excerpt

Table of contents

1.0 INTRODUCTION
1.1 LIMITATIONS OF GENOMICS AND PROTEOMICS STUDIES

2.0 METHODS OF STUDYING PROTEINS
2.1 PROTEIN DETECTION WITH ANTIBODIES (IMMUNOASSAYS)
2.2 ANTIBODY-FREE PROTEIN DETECTION

3.0 CURRENT RESEARCH METHODOLOGIES
3.1 HIGH-THROUGHPUT PROTEOMIC TECHNOLOGIES
3.2 REVERSE-PHASED PROTEIN MICROARRAYS

4.0 PRACTICAL APPLICATIONS OF PROTEOMICS
4.1 INTERACTION PROTEOMICS AND PROTEIN NETWORKS
4.2 PROTEOGENOMICS
4.3 STRUCTURAL PROTEOMICS
4.4 COMPUTATIONAL METHODS IN STUDYING PROTEIN BIOMARKERS

5.0 EMERGING TRENDS IN PROTEOMICS
5.1 PROTEOMICS FOR SYSTEMS BIOLOGY
5.2 HUMAN PLASMA PROTEOME

REFERENCES

Abstract

A huge number of genes within the human genome code for proteins that mediate and/or control genetics processes. Although a large body of information on the number of genes, on chromosomal localisation, gene structure and function has been gathered, we are far from understanding the orchestrated way of how they make metabolism. Nevertheless, based on the genetic information emerging on a daily basis, we are offered fantastic new tools that allow us new insights into the molecular basis of human metabolism under normal as well as pathophysiological conditions. Recent technological advancements have made it possible to analyse simultaneously large sets of mRNA and/or proteins expressed in a biological sample or to define genetic heterogeneity that may be important for the individual response of an organism to changes in its nutritional environment. Applications of the new techniques of genome and proteome analysis are central for the development of nutritional sciences in the next decade and its integration into the rapidly developing era of functional genomics.

Keywords: Genome: Proteome: Genetics research

1.0 INTRODUCTION

Proteomics is the large-scale study of proteins (Anderson N.G et al., 1998 and Blackstock W.P et al., 1999). Proteins are vital parts of living organisms, with many functions. The term proteomics was coined in 1997 (P. James, 1997) in analogy with genomics, the study of the genome. The word proteome is a portmanteau of prote in and gen ome, and was coined by Marc Wilkins in 1994 while he was a PhD student at Macquarie University (Wasinger, 1995). Macquarie University also founded the first dedicated proteomics laboratory in 1995 (Swinbanks, 1995) (the Australian Proteome Analysis Facility – APAF) APAF, 2017.

The proteome is the entire set of proteins that are produced or modified by an organism or system. This varies with time and distinct requirements, or stresses, that a cell or organism undergoes (Anderson et al, 2016).Proteomics is an interdisciplinary domain that has benefitted greatly from the genetic information of the Human Genome Project; (Hood et al., 2013)it also covers emerging scientific research and the exploration of proteomes from the overall level of intracellular protein composition, structure, and its own unique activity patterns. It is an important component of functional genomics.

While proteomics generally refers to the large-scale experimental analysis of proteins, it is often specifically used for protein purification and mass spectrometry. After genomics and transcriptomics, proteomics is the next step in the study of biological systems. It is more complicated than genomics because an organism's genome is more or less constant, whereas the proteome differs from cell to cell and from time to time. Distinct genes are expressed in different cell types, which means that even the basic set of proteins that are produced in a cell needs to be identified.

In the past this phenomenon was done by RNA analysis, but it was found not to correlate with protein content (Simon Rogers, et al., 2008 and VikasDhingraa, et al., 2005). It is now known that mRNA is not always translated into protein (Buckingham, Steven, 2003) and the amount of protein produced for a given amount of mRNA depends on the gene it is transcribed from and on the current physiological state of the cell. Proteomics confirms the presence of the protein and provides a direct measure of the quantity present.

1.1 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.
- as mentioned above many proteins experience post-translational modifications that profoundly affect their activities; for example some proteins are not active until they become phosphorylated. Methods such as phosphoproteomics and glycoproteomics are used to study post-translational modifications.
- many transcripts give rise to more than one protein, through alternative splicing or alternative post-translational modifications.
- many proteins form complexes with other proteins or RNA molecules, and only function in the presence of these other molecules.
- protein degradation rate plays an important role in protein content (Archana Belle, 2006).

2.0 METHODS OF STUDYING PROTEINS

In proteomics, there are multiple methods to study proteins. Generally, proteins can either be detected using antibodies (immunoassays) or using mass spectrometry. If a complex biological sample is analyzed, either a very specific antibody needs to be used in quantitative dot blot analysis (qdb), or then biochemical separation needs to be used before the detection step as there are too many analytes in the sample to perform accurate detection and quantification.

2.1 PROTEIN DETECTION WITH ANTIBODIES (IMMUNOASSAYS)

Antibodies to particular proteins or to their modified forms have been used in biochemistry and cell biology studies. These are among the most common tools used by molecular biologists today. There are several specific techniques and protocols that use antibodies for protein detection. The enzyme-linked immunosorbent assay (ELISA) has been used for decades to detect and quantitatively measure proteins in samples. The Western blot can be used for detection and quantification of individual proteins, where in an initial step a complex protein mixture is separated using SDS-PAGE and then the protein of interest is identified using an antibody.

Modified proteins can be studied by developing an antibody specific to that modification. For example, there are antibodies that only recognize certain proteins when they are tyrosine-phosphorylated, known as phospho-specific antibodies. Also, there are antibodies specific to other modifications. These can be used to determine the set of proteins that have undergone the modification of interest.

Disease detection at the molecular level is driving the emerging revolution of early diagnosis and treatment. A challenge facing the field is that protein biomarkers for early diagnosis can be present in very low abundance. The lower limit of detection with conventional immunoassay technology is the upper femtomolar range (10(-13) M). Digital immunoassay technology has improved detection sensitivity three logs, to the attomolar range (10(-16) M). This capability has the potential to open new advances in diagnostics and therapeutics, but such technologies have been relegated to manual procedures that are not well suited for efficient routine use (Wilson, DH, et al., 2016).

2.2 ANTIBODY-FREE PROTEIN DETECTION

While protein detection with antibodies are still very common in molecular biology, also other methods have been developed that do not rely on an antibody. These methods offer various advantages, for instance they are often able to determine the sequence of a protein or peptide, they may have higher throughput than antibody-based and they sometimes can identify and quantify proteins for which no antibody exists.

3.0 CURRENT RESEARCH METHODOLOGIES

Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE) (Tonge R, et al., 2001) can be used to quantify variation in the 2-D DIGE process and establish statistically valid thresholds for assigning quantitative changes between samples (Tonge R, et al., 2001). Comparative proteomic analysis can reveal the role of proteins in complex biological systems, including reproduction. For example, treatment with the insecticide triazophos causes an increase in the content of brown planthopper (Nilaparvatalugens (Stål)) male accessory gland proteins (Acps) that can be transferred to females via mating, causing an increase in fecundity (i.e. birth rate) of females (Li-Ping Wang, et al., 2010). To identify changes in the types of accessory gland proteins (Acps) and reproductive proteins that mated female planthoppers received from male planthoppers, researchers conducted a comparative proteomic analysis of mated N. lugens females (Ge, Lin-Quan et al., 2011). The results indicated that these proteins participate in the reproductive process of N. lugens adult females and males (Ge, Lin-Quan, et al., 2011)

Proteome analysis of Arabidopsis peroxisomes (Reumann S, 2011) has been established as the major unbiased approach for identifying new peroxisomal proteins on a large scale (Reumann S, 2011)

There are many approaches to characterizing the human proteome, which is estimated to contain between 20,000 and 25,000 non-redundant proteins. The number of unique protein species will likely increase by between 50,000 and 500,000 due to RNA splicing and proteolysis events, and when post-translational modification are also considered, the total number of unique human proteins is estimated to range in the low millions.(Uhlen M, et al., 2005 and Ole Nørregaard Jensen, 2004).

In addition, the first promising attempts to decipher the proteome of animal tumors have recently been reported. (Klopfleisch R, et al., 2010).

3.1 HIGH-THROUGHPUT 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.

3.1.1 Mass spectrometry and protein profiling

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.

The second quantitative approach uses stable isotope tags to differentially label proteins from two different complex mixtures. Here, the proteins within a complex mixture are labeled first isotopically, and then digested to yield labeled peptides. The labeled mixtures are then combined, the peptides separated by multidimensional liquid chromatography and analyzed by tandem mass spectrometry. Isotope coded affinity tag (ICAT) reagents are the widely used isotope tags. In this method, the cysteine residues of proteins get covalently attached to the ICAT reagent, thereby reducing the complexity of the mixtures omitting the non-cysteine residues.

Quantitative proteomics using stable isotopic tagging is an increasingly useful tool in modern development. Firstly, chemical reactions have been used to introduce tags into specific sites or proteins for the purpose of probing specific protein functionalities. The isolation of phosphorylated peptides has been achieved using isotopic labeling and selective chemistries to capture the fraction of protein among the complex mixture. Secondly, the ICAT technology was used to differentiate between partially purified or purified macromolecular complexes such as large RNA polymerase II pre-initiation complex and the proteins complexed with yeast transcription factor. Thirdly, ICAT labeling was recently combined with chromatin isolation to identify and quantify chromatin-associated proteins. Finally ICAT reagents are useful for proteomic profiling of cellular organelles and specific cellular fractions (Weston et al., 2004).

Another quantitative approach is the Accurate Mass and Time (AMT) tag approach developed by Richard D. Smith and coworkers at Pacific Northwest National Laboratory. In this approach, increased throughput and sensitivity is achieved by avoiding the need for tandem mass spectrometry, and making use of precisely determined separation time information and highly accurate mass determinations for peptide and protein identifications.

3.1.2 Protein chips

Balancing 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. The first version of such arrays consisted of 5000 purified proteins from yeast deposited onto glass microscopic slides. Despite the success of first chip, it was a greater challenge for protein arrays to be implemented. Proteins are inherently much more difficult to work with than DNA. They have a broad dynamic range, are less stable than DNA and their structure is difficult to preserve on glass slides, though they are essential for most assays. The global ICAT technology has striking advantages over protein chip technologies.

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Details

Title
Proteomics. Importance for the Future of Genetics Research
College
University of Lagos  (University of Lagos)
Course
Cell Biology and Genetics
Grade
14.0
Author
Year
2020
Pages
19
Catalog Number
V517924
ISBN (eBook)
9783346120106
ISBN (Book)
9783346120113
Language
English
Tags
Proteomic, genomic
Quote paper
Kehinde Sowunmi (Author), 2020, Proteomics. Importance for the Future of Genetics Research, Munich, GRIN Verlag, https://www.grin.com/document/517924

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