Proteomics

Tracing the footprints of Proteomics To comp atomic number 18 and instruction the techniques used in proteomics since the last decade. Abstract Proteomics is a oeuvre of the proteome of an organism. The last few decades ache seen a rapid gain in the development of this field. This root word attempts to compare and contrast the way in which proteomics studies are performed today as opposed to those performed ten days ago and analyse its future implications. The thrust of research while studying biology at a molecular level initially was focused specifically on the genomes of various organisms.As scientists disc everyplaceed the intricacies of genes and their functionalities, the care was soon drawn towards the end result of the central dogma of molecular biology, namely, the proteins, produced through translation of RNAs. Therefore, to study the proteins produced in an organism, referred to as the proteome, not just as products of a genome, but to a greater extent importantly how they interact and bring ab pop changes at the macro level, the field of proteomics has emerged. (1)Proteins play a pivotal role in carrying out various functions in a body at the structural and dynamic levels. Proteins as enzymes and horm hotshots nonplus the vital metabolic processes and as structural components provide stability to the cellphoneular components. The knowledge obtained through the study of these systems confines an insight into the overall functioning of the living organisms. In spite of having similar genetic blue prints, the protein building in various organisms are regulated differently through diverse ne iirks of protein-protein interactions.Hence, proteomics provides an understanding about these regulatory processes and establishes the differences and similarities amongst the evolutionary pathways of the organisms by grouping them under phylogentic trees. Further, drugs can be real for specific diseases by designing structural analogues of proteins re sponsible for diseased conditions after elucidating their structures, which can then up or subdue regulate metabolic processes.Thus, the study of proteins plays an essential part of researches carried out in other related palm of study such as developmental and evolutionary biology and drug designing. (1)(2) Since the invention of the 2-Dimentional Gel dielectrolysis in the 1970s, which is considered to be the stepping stone of modern day protein studies, scientists have been constant quantityly striving to develop mod and potent methods to study proteomics.Thus, this paper is an attempt to identify and compare these techniques which have been used and improved over the last decade. The popular and preferred procedure to study the proteome of an organism comprises of three major travel, isolation, detachment on 2-D mousse and analysis through a mass spectrometer. Most of the improvements revolve around this basic protocol. 2-D change electrophoresis was one of the first meth ods which were used to analyse the proteome of an organism. In this technique, the protein is scattered on the basis of its military mission and size.The proteins are first separated on the basis of their different charges in the 1st dimension, following which they are separated on the 2nd dimension on the basis of their molecular weight. The change or social occasion provides a graphical representation of each protein after musical interval and hence they can be secernate individually. However, the reproducibility of the results obtained through such an analysis has not been satisfactory. Till date there are constant efforts being made to improve the efficacy of this technique, such that a bouffant number of proteins could be separated at the same time.The first 2-D separation which was carried out by using the electrophoresis buffer and amylum gel, the improvements which followed gave rise to the foundation of modern day 2-D separation, which was combining two 1-d techn iques involving separation on the basis of pH using isoelectric focusing (IEF) and using SDS-Page for separation on the basis of molecular weight after the samples have been prepared specifically using various reagents such as Urea (as a chaotrope to solubilise) and DTT (to break di-sulphide linkages without fragmentation into peptides), in a suitable buffer (3).Further, for certain segments of proteins which were hydrophobic in nature, like those found in the cell membrane, it was discovered that special reagents such as thiourea, sulfobetaine and tributyl phosphine which are classified as chaotropes, surfactants and reducing agents respectively, back up their solubility during sample preparation in the lead running them on the gel. Another notable file name extension of 2-D separation was the use of IPG strips, which had different pH gradients. These strips were made available commercially and drastically contributed to the convenience of the technique.Also, experiments were c arried out using a number of such strips to increase the range of pH, hence successfully accommodating a large number of proteins(4). Nevertheless, such a method, although successful, was human-error prone and hence the results on the varied from each other in majority of cases. To overcome this, a number of replicates of the gel had to be prepared and therefore demanded a bevy of labour. To overcome this barrier, the differential gel electrophoresis technique DIGE was developed. In this method, the proteins are labelled with fluorescent dyes antecedent to electrophoresis.The fluorophores are joined via an amide linkage to the amino acid lysine and therefore the proteins can be resolved in concert on the same gel through distinguished patterns of fluorescent emissions (5). Further advancement of the standard 2-D gel analysis was to incorporate automation to the technology, however the room for automation to analyse the results was limited due to the inability of a computer to dis tinguish between the different patterns. Differentiating a spot of protein on a gel, its bulk and to separate it from a background console remains an overwhelming task for the computer.The next step in proteome analysis is protein identification using mass spectrometry (MS). One of the most compelling problems of using MS to study biomolecules such as proteins was the inability to obtain ions of sufficiently large size which would utilely three to their identification. Since the development of Electron Spray ionization and MALDI (Matrix assisted Laser Desorption Ionization) this drawback of MS was overcome and today the combining of these ion sources with different mass analysers e. g.MALDI-TOF/TOF, ESI Q-TOF and ESI triple quardrupoles are used widely in proteomics. Identification of a protein is carried out through a process referred to as peptide mass fingerprinting (PMF). In this technique, proteins that have been separated on a 2-D gel are excised and digested into peptides using proteases such as trypsin. The digested peptides, when subjected to study in a MS, give a characteristic m/z spectrum. The protein can be indentified when this data correlates to the data in protein databases compared using softwares base specific algorithms.However, to extrapolate a proteins role in metabolism, it is overly undeniable to identify how the protein is modified after translation. Post translation modification plays an important role in acting like a regulating switch modifications such as phosphorylation play an important in processes such as cell signalling. The main drawback while analysing a phosphorylated protein through MS was its signal suppression. To rectify this issue, high performance separation techniques such as HPLC were conjugated with the MS LC-MALDI-MS is an example of such a combination (6).Further extension of the protein mass fingerprinting was the development of shotgun proteomics, to specifically do away with the dis rewards of a standar d 2-D gel analysis. This technique is based on separation of peptides obtained after protease digestion, using multidimensional chromatography. It is necessary that the two dimension of this multidimensional separation done using chromatography are orthogonal in nature, i. e. using two different properties of a protein similar to a 2-D gel separation which uses pI and mass.Separating proteins using reversed phase, based on hydrophobicity, and Strong cation exchange, using the charged state of the peptides is an example of separation in two dimensions. Although the PMF feeler provided a successful identification process to recognize the proteins present in a proteome, it was also necessary to study the changes in protein expression in response to a stimulus. To achieve this, the technique call the ICAT was developed which protein mixtures from after isolation were modified such that they can differentiated on the basis of mass from one cellular location to another.In ICAT, this modi fication is done using a cysteine with an isotope labelled biotin tag. Today, the efforts to develop mod technologies are directed towards automation in sample preparation and effective interfacing with other techniques. Interfacing has been achieved more than successfully with ESI than MALDI owing to its ability of operating with a continuous flow of liquid (7). Sample from organisms curb thousands of proteins, to effectively separate certain important proteins such as disease biomarkers from this mixture, is a highly demanding task.Further, effective proteolytic digestion can be challenging when the proteins of interest are present in low quantities. Therefore, before a sample of protein can be effectively analysed there are a number of steps to be performed which are prone to human error and are laborious. The development of Micro-fluidic system as an larboard with the mass spectrometer such as ESI provides the option of automating this process and hence making proteome analy sis more effective less time-consuming.Therefore, such a chip based technology has a clear advantage over the traditionally used methods due its improved probability of obtaining the protein of interest, reduced consumption of reagents and accelerated chemical reaction time. The micro fluidic chips can be directly coupled to an ESI- MS using a pressure compulsive or electro-osmotic flow. Thus, such a system where there is a direct interface is called an on-line setup. On the other hand, such a setup cannot be achieved in MALDI where a mechanical bridge is created between the micro-fluidic chip and the Mass spectrometer.The first step of a proteome analysis, i. e. sample purification is carried out using a hydrophobic membrane integrated into an inlet channel of a polyimide chip. Separation of proteins from the sample can be achieved either using a capillary electrophoresis (CE) or a liquid chromatographic (LC) method. CE is usually preferred over LC due as it provides a faster sepa ration and can be coupled to an electric pump. Proteolytic digestion is carried out on the solid surface of the chips, where the enzymes are immobilized.Thus, such a chip provides a platform for the automation of the initial steps of a proteomic study, and more studies are still being performed to increase the efficacy of this approach (8). To conclude, over the last decade, there has been a rapid progress in the techniques used to study proteomics. The direction of progress has also shed a light on the importance of proteomics and the implications if would have in the coming years. Studies on evolution have benefitted a great commode with the development of techniques like ICAT which enhances quantitative and proportional studies of the different proteomes.In the field of medicine and drug discovery, the masking of these techniques, paves the road for discovery of novel biomarkers for specific diseases in a quicker and less complicated manner. Further, it would also assist vacci ne development by identifying specific antigens for a disease. The developments of micro-fluidic chips have opened the door for new diagnostics techniques by characterizing effectively the protein responsible for a diseased state. Such an approach has already been apply to study the proteins produced in the body in a cancerous state.Therefore, as more researchers and academics adapt these with these applications, some more improvements would soon evolve. References 1. Anderson, L. , Matheson, A. and Steiner, S. (2000). Proteomics applications in basic and applied biology. Current Opinion in Biotechnology Vol 11pp. 408412. 2. Pazos, F. and Valencia, A. (2001). Similarity of phylogenetic trees as indicator of protein protein interaction. Protein Engineering Vol 14 no 9 pp. 609-614. 3. Klose, J. (2009). From 2-D electrophoresis to proteomics. dielectrolysis Vol 30 pp. 142149. 4. Herbert, B. (1999). Advances in protein solubilisation for two-dimensional electrophoresis. Electroph oresis Vol 20 pp. 660- 663. 5. Alban, A. , David, S. , Bjorkesten, L. , Andersson, C. , Sloge, E. , Lewis, S. and Currie, I. (2003). A novel experimental design for comparative two-dimensional gel analysis Two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics Vol 3 pp. 3644. 6. Reinders, J. , Lewandrowski, U. , Moebius, J. , Wagner, Y. and Sickmann, A. (2004). Challenges in mass spectrometry based proteomics. Proteomics Vol 4 pp. 36863703. 7. Swanson, S. and Washburn, M. (2005). The continuing evolution of shotgun proteomics. medicate Discovery Today Vol 10. 8. Lee, J. , Sopera, S. and Murraya, K. (2009). Microfluidic chips for mass spectrometry-based proteomics. Journal of Mass Spectrometry Vol 44 pp. 579593.