Proteomics: A Powerful Tool for Biometric Scientists

Proteomics is the study of proteomes on a large scale. A proteome is a collection of proteins made by an organism, system, or biological context. We can refer to a species' proteome (for example, Homo sapiens) or an organ's proteome (for example, the liver). 

 

The proteome is not constant; it varies between cells and changes over time. To some extent, the proteome reflects the transcriptome. However, protein activity (often measured by the rate of reaction of the processes in which the protein is involved) is influenced by a variety of factors in addition to the level of expression of the relevant gene. Lets understand this in detail in the blog below! 

 

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What is Proteomics?

 

The term "proteomics" refers to the systematic study of proteins. It works in tandem with other "omics" technologies such as genomics and transcriptomics to determine the identity and function of an organism's proteins. 

 

Proteomics is used in many areas of research, including the identification of diagnostic markers and vaccine candidates, the study of pathogenic mechanisms, the analysis of expression patterns at different time points and in response to different stimuli, and the elucidation of functional protein networks. 

 

Sample preparation, protein separation, and protein identification are all part of the proteomics analysis process. Proteomics is critical for early disease diagnosis, prognosis, and disease progression monitoring. It also plays an important role in drug development as target molecules. 

 

Proteomics is the study of the proteome, which includes the expression, structure, functions, interactions, and modifications of proteins at any stage. The proteome changes over time, from cell to cell, and in response to external stimuli. Proteomics in eukaryotic cells is complicated due to post-translational modifications that occur at various sites in a variety of ways.

 

Although much more complex than genomic, proteomics is one of the most important methodologies for understanding gene function. Fluctuations in gene expression levels can be detected using transcriptome or proteome analysis to distinguish between two biological states of the cell. 

 

Microarray chips have been developed for large-scale transcriptome analysis. However, microarrays cannot directly measure increased mRNA synthesis. Proteins are biological function effectors, and their levels are influenced not only by corresponding mRNA levels, but also by host translational control and regulation. 

 

As a result, proteomics would be regarded as the most pertinent data set for characterizing a biological system. Proteomics is used to look into:

 

1. When and where protein expression occurs

 

2. Protein production, degradation, and steady-state abundance rates

 

3. How proteins are modified (for example, phosphorylation and other post-translational modifications (PTMs))

 

4. Protein transport between subcellular compartments

 

5. Protein involvement in metabolic pathways

 

6. The manner in which proteins interact with one another

 

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Importance of Proteomics

 

The human genome contains all of the genes needed to create a fully functional human being. However, the genome is only a repository of data. It must be expressed in order to function.

 

The first stage of gene expression is transcription, which is followed by messenger RNA translation to produce proteins. The proteome is the entire set of proteins produced by the genome at any given point in time.

 

The proteome is also constantly changing. Regardless of cell type, developmental stage, or environmental conditions, the majority of our cells have the same genome. However, the proteome varies significantly in these different circumstances due to different patterns of gene expression and protein modification.

 

Proteomics, or proteome research, is important because proteins are the actual functional molecules in the cell. When DNA mutations occur, it is the proteins that are ultimately impacted. 

 

When drugs have beneficial effects, they do so by interacting with proteins. Proteomics thus encompasses a variety of aspects of protein function, including the following:

 

1. Structural proteomics is the study of protein structures on a large scale. Comparing protein structures can aid in determining the functions of newly discovered genes. 

 

In addition, structural analysis can reveal where drugs bind to proteins and where proteins interact with one another. This is accomplished through the use of technologies such as X-ray crystallography and NMR spectroscopy.

 

2. Expression Proteomics can aid in identifying the main proteins found in a specific sample as well as proteins that differ in expression in related samples, such as diseased vs healthy tissue. A protein found only in diseased samples could be a promising drug target or diagnostic marker. Proteins with similar expression profiles may be functionally related as well. 2D-PAGE and mass spectrometry are used in this study.

 

3. Interaction proteomics is the study of protein interactions on a large scale.

 

Protein-protein interactions can be used to determine protein functions and to show how proteins assemble in larger complexes. Affinity purification, mass spectrometry, and the yeast two-hybrid system are all particularly useful.

 

Proteome analysis techniques are not as simple as those used in transcriptomics. The advantage of proteomics is that it studies the cell's actual functional molecules. Strong gene expression, which results in abundant mRNA, does not always imply that the corresponding protein is also abundant or active in the cell.

 

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Techniques of Proteomics

 

The techniques for proteomics can be further divided into 2 main categories :- 

 

Based on Chromatography

 

1. Chromatography based on ion exchange

 

The IEC is a versatile tool for protein purification based on charged groups on its surface. The amino-acid sequences of the proteins differ; some amino acids are anionic, while others are cationic. Equilibrium between these charges determines the net charged content of a protein at physiological pH. 

 

It first separates the proteins based on their charge nature (anionic and cationic), then on their comparative charge strength. Because of its low cost and ability to persist in buffer conditions, the IEC is extremely valuable.

 

The Neutrophil Activator Protein (HP-NAP) is a Helicobacter pylori virulence factor that can activate human neutrophils by secreting mediators and reactive oxygen species. The HP-NAP is a potential H. pylori diagnostic marker, as well as a potential drug target and vaccine candidate. 

 

Shih et al. used one step anionic exchange chromatography to purify the recombinant HP-NAP expressed in B. subtilis with 91 percent recovery. Mussel adhesive proteins (MAPs) have unique biocompatible and adhesive properties that can be used in biomedical and tissue engineering applications.

 

2. Chromatography by size exclusion

 

SEC separates proteins based on permeation through a porous carrier matrix with distinct pore size; thus, proteins are separated based on molecular size. The SEC is a robust technique capable of handling proteins in a variety of physiological conditions, including the presence of detergents, ions, and co-factors, as well as at different temperatures. 

 

The SEC is a powerful tool for purifying non-covalent multimeric protein complexes under biological conditions and is used to separate low molecular weight proteins.

 

Trichomonas vaginalis soluble factors have the ability to damage target cells and are involved in the pathogenesis of trichomoniasis. The phospholipase A2-like lytic factor was purified, and further characterization revealed two fractions of 168 and 144 kDa.

 

SEC was used to purify antimicrobial peptides synthesized by the marine bacterium Pseodoalteromonas from culture supernatant. These peptides have a strong inhibitory effect against pathogens involved in skin infections. 

 

Arabidopsis thaliana cytosolic proteins have been purified in order to better understand how the cell coordinates various mechanical, metabolic, and developmental activities. SEC was also used to purify intrinsically disordered proteins from A. 

 

3. Chromatography of Affinity

 

Affinity chromatography was a significant breakthrough in protein purification, allowing researchers to investigate protein degradation, post-translational modifications, and protein-protein interactions. 

 

The reversible interaction between the affinity ligand of the chromatographic matrix and the proteins to be purified is the basic principle underlying affinity chromatography 

 

Affinity chromatography has a wide range of applications in identifying microbial enzymes that are primarily involved in pathogenesis. Metal chelate affinity chromatography was used to rapidly purify the homodimer and heterodimer of HIV-I reverse transcriptase.

 

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Advanced Methods

 

1. Microarray of proteins

 

Protein microarrays, also known as protein chips, are a new class of proteomics techniques that can detect high-throughput signals from small amounts of samples. Analytical protein microarrays, functional protein microarrays, and reverse-phase protein microarrays are the three types of protein microarrays.

 

2. Protein microarray analysis

 

The most common type of analytical protein microarray is antibody microarray. Direct protein labeling is used to detect proteins after antibody capture. These are commonly used to assess protein expression levels and binding affinities. 

 

A high-throughput proteome analysis of cancer cells was performed using antibody microarray for differential protein expression in tissues derived from oral cavity squamous carcinoma cells. Protein profiling of bladder cancer was also accomplished using an antibody array.

 

3. Microarray of Functional Proteins

 

Because functional protein microarrays are made from purified protein, they can be used to study various interactions such as protein-DNA, protein-RNA, protein-protein, protein-drug, protein-lipid, and enzyme-substrate relationships. 

 

The first application of functional protein microarray was to investigate the substrate specificity of yeast protein kinases . Thousands of proteins' functions were characterized using a functional protein microarray.

 

4. Protein Microarray in Reverse Phase

 

Cell lysates from various cell states are arrayed on nitrocellulose slides, which are then probed with antibodies against target proteins. Antibodies are then detected using fluorescent, chemiluminescent, and colorimetric assays. Reference peptides are printed on slides for protein quantification. 

 

These microarrays are used to identify the altered or dysfunctional protein that is associated with a specific disease. The reverse-phase protein microarray analysis of hematopoietic stem cell and primary leukemia samples was found to be highly reproducible and reliable for large-scale analysis of phosphorylation state and protein expression in human stem cells and acute myelogenous leukemia cells.

 

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Types of Proteomics

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Types of Proteomics

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The major types of Proteomics are :-

 

1. Structural Proteomics

 

Structural Proteomics refers to proteomics studies that aim to map out the proteins present in a specific cellular organelle or the structure of protein complexes. Structural analysis can help identify the functions of newly discovered genes, as well as where drugs bind to proteins and where proteins interact with one another. 

 

X-ray crystallography and nuclear magnetic resonance spectroscopy are two technologies used in structural proteomics. Expression proteomics is the quantitative study of protein expression differences between samples that differ by a specific variable. 

 

This type of proteomics can help identify the main proteins found in a specific sample as well as proteins that are expressed differently in related samples, such as when comparing diseased and healthy tissue. 2D-PAGE and mass spectrometry are used in this study.

 

2. Functional Proteomics

 

Functional proteomics is a broad term for a variety of targeted proteomics methodologies. Protein-protein interactions are characterized to determine protein functions and to demonstrate how proteins assemble in larger complexes. In some cases, affinity chromatography is used to isolate specific subproteomes for further analysis. 

 

Proteomics is the study of proteins on a large scale. Proteins are essential components of living organisms, performing numerous functions such as the formation of structural fibers in muscle tissue, enzymatic digestion of food, and DNA synthesis and replication. 

 

Other types of proteins include antibodies, which protect an organism from infection, and hormones, which send vital signals throughout the body. The proteome is the entire set of proteins that an organism or system produces or modifies. 

 

Proteomics allows for the identification of a growing number of proteins. This varies with time and the specific needs, or stresses, that a cell or organism faces.