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Proteomics - New biology in the field of Science
#1
In the last decade, there has been vast development in the field of science and biomedicine. The whole sequencing of the human genome is certainly a milestone in the development and progress of science. This has helped in the rapid development in the field of medicine by providing knowledge about gene therapy, individual-based treatment for various diseases, different modes of treatment for genetic diseases, etc. Proteome can be defined as the set of various proteins expressed by a particular genome. The study of the different proteins, their dynamics and their interplay and interactions constitute the study of proteomics.

Although, both Proteomics and Protein chemistry involve the identification of various proteins expressed within the body, they are totally unrelated to each other. The protein chemistry is a part of the structural biology, dealing with physical biochemistry of the various proteins from a mechanistic point of view and relates mainly to the structure and function modelling studies and the effect of one on the other. Proteomics, on the other hand, involves systems biology, dealing with multiprotein systems and characterizing the behaviour of the whole system.

Measurement of gene expression is possible using various techniques; however, study of proteomics plays an important role considering the instability of the mRNAs preventing the formation of proteins as well as the regulatory functions of the different proteins in the biological and molecular mechanisms within the body. The gene expression can be measured by the use of cDNA as well as microarray system. Analysis of proteome is a complex task owing to the presence of posttranslational modification in proteins and also the fact that protein recognition is not based on sequence unlike oligonucleotides. Hence, special tools have been developed for proteomics such as

i)databases of protein, EST and genome sequence Mass spectrometry (MS),
ii)Mass Spectrometry (MS)
iii)collection of software that is capable of comparing the MS data with the protein sequence database, and
iv)protein separation technology

Analytical proteomics has become an emerging field in science. The identification of different proteins within the cell and their characterization is gaining importance even in medical field. The analysis of the whole protein is somewhat difficult. Hence, the tools for proteomics utilize different approaches for proper protein analysis. The protein is firstly converted to peptides and the sequence of the peptides is analysed. The sequence of the peptides is then matched with the sequence in the database to identify the proteins. The main problem in proteomics is the presence of a protein mixture in the biological sample. The protein analysis with the use of mixture is difficult; hence, the separation of the proteins is essential. The separation techniques involve the use of SDS-PAGE, which may be one-dimensional (1D) or two-dimensional (2D), High Performance Liquid Chromatography (HPLC). Proteins have also been analysed by digesting them and then carrying out separation using capillary electrophoresis or Isoelectric focusing (IEF). However, the protein separation by 2D SDS-PAGE followed by digestion into peptide fragments has emerged to be the better approach in analytical proteomics, due to numerous advantages offered by 2D SDS-PAGE. The proteins are digested using various proteases, which cleave at specific amino acids, thereby helping in the analysis with MS. Two types of instruments have been used for proteomics study: the MALDI-TOF and the ESI-tandem MS. Although, both the instruments work on completely different principles, they provide complementary information, hence both serve definite unique purpose. Peptide mass fingerprinting is a protein identification method used in high throughput proteomic study. In this method, the proteins are digested using trypsin and mass is analysed using MALDI-TOF. However, the difficulty in differentiation of homologous proteins and the similar proteins from different species limit the use of the method.

The main four applications of proteomics are:- Mining, protein expression profiling, protein network mapping and mapping of protein modifications. Mining refers to the identification of the proteins in a given sample i.e. the composition of the proteome is identified from the gene expression data from microarrays. Protein expression profiling is the identification of protein composition specific for a particular state of an organism or as a function to the effect of a drug or any other stimulus. Protein network mapping refers to the study of the interactions of various proteins in the functional network within the body and gives detailed information about the proteins in any signal transduction pathway. Mapping of protein modifications involves the study and identification of the nature and specificity of the posttranslational modification in a given protein. The development of protein arrays is under progress and if found successful, will create a great impact in the field of proteomics.
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#2
Application of the proteomics

Genome is entire set of genes that one organism can express while proteome is set of proteins that one organism can produce. Since all PROTEins are expressed from the genOME - name of the scientific discipline focused on protein examination and discovery was easily coined. Proteomics is more complex field compared to genomics; each cell contains same set of genes, but they will express differently depending on the cells type (protein expression is tissue/organ specific), developmental stage (from embryo to adult stage), environmental effects….

Expression of 20,000 – 25,000 genes results in ~ 1,000,000 functional proteins in the human body. Alternative splicing and post-translational modifications increase both protein number and their diversity. Disagreement in number of available genes and end product proteins suggests that genomics can’t provide all necessary info in the protein discovery. That’s why proteomics is so important. Proteins play multiple roles in our body: they regulate genetic expression, induce immune response (antibodies), allow muscles contraction, transport various larger or smaller molecules, act like hormones, accelerate biochemical reactions (enzymes)… Focused discovery and characterization of proteins makes proteomics applicable to many scientific fields and provide a lot of medical solutions.

Proteomics in biomarker discovery

Biomarkers are all molecules (genes, proteins, hormones…) that could indicate specific physiological or pathological process. Increased level of antibodies indicates infection, elevated AST or ALT serum levels could suggest liver damage, beta-hCG (both in serum and urine) is early marker of pregnancy… In medical field they are important for disease prevention and for early diagnostics. Pharmaceutical industry could accelerate drug discovery process by narrowing selection of potential candidates based on drug target identification and drug response (drug efficacy and/or toxicity) in organism. Most biomarker discovery experiments are applying mass spectral-based proteomic technologies using biological samples of unknown protein quantity. Success in biomarker discovery always depends on the quality of biological sample, ability to determine precise protein level and in correct interpretation of the collected data.

Proteomics in the study of the cancer metastasis

Tumor metastasis is the leading cause of death in cancer patients. Metastasis could spread locally (in the same organ where cancer is generated) or travel to a distant part of the body via lymphatic and vascular system, resulting in metastatic (secondary) tumor formation. Most commonly, secondary tumors are detected in the brain, lungs, bones and liver. Routes of cancer spreading are well studied, but molecular and cellular mechanisms of metastasis are still poorly identified. Proteomics is focused on identification of the proteins associated with metastatic process with the main goal to facilitate clinical management and improve therapeutic strategies necessary for metastasis prevention.

Proteomics in neurology

Neurodegeneration can be described as loss of structure and function of neural cells. Genetic mutation can be a trigger for different neurodegenerative disorders. For example, CAG nucleotide triplet repeats (encoding amino acid glutamine) are common feature in polyglutamine diseases such as Huntington’s disease or spinocerebellar ataxia. Protein misfolding and its aggregation is common feature in neurodegenerative proteopathies such as Parkinson’s (result of alpha synuclein aggregation) and Alzheimer’s disease (alpha synuclein, tau and beta amiloid aggregation). Also, altered protein degradation pathways could result in neurodegenerative disorder. Toxic accumulation of proteins in cytosol is typical for Parkinson’s and Huntington’s disease, in nucleus for spinalcerebellar ataxia, in endoplasmatic reticulum for familial encephalopathy with neuroserpin inclusion bodies, and extracellulary for Alzheimer’s disease. Neurodegenerative disorders are associated with impaired cognition, reduced mobility (neuro-muscular impairments), speech difficulties, pain…. Most commonly applied techniques for protein analyses in this field are two-dimensional gel electrophoresis while mass spectrometry is used for protein identification. Current therapeutic approaches could be improved by revealing pathophysiological protein networks in neural tissue. Proteomics could be of great value by discovering enzymes, cytoskeleton, synaptosomal and antioxidant proteins implicated in disease genesis.

Proteomics in cardiovascular disorders

All disorders that are affecting hearth and vascular system are known as cardiovascular disorders. This is large and diverse class of pathologies triggered (usually) by atherosclerosis and hypertension. Cardiovascular disorders are very common today, affecting mostly older people. Cardiovascular impairment is the number one cause of death in the modern society. Proteomics is used to identify modified proteins and delicate processes in cardiac and vascular tissue during development and progression of the disease. Novel insight in pathological processes could improve therapeutics and reduce mortality rate.

Proteomics can be applied in many other (important) fields, from antibody profiling, diabetes and nutrition research to fetal and maternal medicine.
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#3
Clinical Proteomics:

The detailed information of the composition and activities of urine proteins could offer novel insights into normal renal physiology. Although proteomic studies have identified a large number of urinary proteins their practical significance remains largely unknown. Furthermore, many proteomic studies do not give explanation for post-translational modifications which may have a important impact on protein function. Many proteins are enzymes that are maintained in a latent state until their activity is compulsory. This allows for rapid host responses, without the time lag required for transcription and translation. Thus, there can be marked changes in functional states in the absence of significant alteration in concentration. These functional changes in activity are undetectable with methods that simply quantify transcript or protein levels, but are significant for characterizing the dynamic physiological standing of the host.

The activity-based protein profiling (ABPP) is a narrative move toward the assess the functional status of selected enzymes in the proteome. Activity-based protein profiling is based on the use of tagged probes that selectively respond with the active sites of a given enzyme or family of enzymes. Activity-based probes consist of a reactive group that aims the active residue of the enzyme, a short linker and reporter tag. The central premise of ABPP is that accessibility of substrate to the active site of an enzyme is an indicator of enzyme activation. Because the underlying molecular mechanisms of catalysis by members of an enzyme family are habitually identical it is possible to develop a single probe to detect the active forms of members from a given family. Members of the serine hydrolase family contribute to a serine centric charge relay system in their catalytic site and this common feature can be exploited with an activity-based probe to selectively label active serine hydrolases. Furthermore, probe-labeled enzymes can be affinity-purified through their tag and identified by mass spectrometry to determine the specific active enzymes within a biological sample.

The serine hydrolase family is one of the major enzyme classes in humans and constitutes ~1-2% of predicted protein products from the eukaryotic genome. Serine hydrolases consist of greater than 100 serine proteases and approximately 110 esterases, lipases, peptidases and amidases. While some members are well-studied (e.g. trypsin, elastase, thrombin, acetylcholinesterase), many have yet to be explained. Indeed the role for ~50% of the non-serine proteases remains undetermined, and very little is known about the occurrence and role of serine hydrolases in the urine of healthy human.
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Proteomics - New biology in the field of Science00