Analysis of Proteomics Problems - Assignment Example

Proteomics Problems Q1. Identifying Methodology for a Phosphoprotein of Interest Proteins are ed to post translational modifications like phosphorylation (addition of phosphate groups) after being transcribed and translated from a genome. Phosphorylation influences the function of phosphoprotein in relation to signal pathways and enzymatic activity. Gel Electrophoresis is the most common method applied to identify phosphoproteins. Typically a two dimensional gel electrophoresis is widely used to identify proteins as it provides high resolution between two nearly identical proteins. To differentiate the phosphoprotein from various other proteins, immune-staining methods using antibodies and radio-labeling methods are applied. The protein separated by electrophoresis is immobilized on hydrophobic membrane and is brought in close proximity to the antibody and the phosphoprotein acts as its target antigen which forms a antigen-antibody complex by any type of antigen –antibody interactions. A second marker antibody is used to detect such antigen-antibody complex (Butler, 1986). With this background there can be a methodology developed for identifying a phosphoprotein of importance. The philosophy is to subject the sample protein for a two dimensional electrophoresis, then dephosphorylating the sample phosphoprotein with phosphatase (phosphatase will cleave the phosphate groups from the protein); then performing the two-dimensional electrophoresis under similar conditions and then detecting the various spots that move through the alkaline gradient and gets iso-electrically focused after being transformed into a zwitterions. The detailed method that can be applied to identify the phosphoprotein can be explained by the following steps. a) The sample proteins need to be solubilized at first. b) Preservation buffers should be added to the sample protein solution for the prevention of precipitation of the proteins and deactivation of the phosphatase. c) The sample protein needs to divided and separated into two groups like X and Y. d) the sample Y to be dephosphorylated with phosphatase. The phosphatase could be recombinant lambda protein phosphatase. Sample Y and the phosphatase mixture should be warmed at 30 degree centigrade for at least an hour. The sample prepared in this way should be subjected to concentrated form by using ultra free centrifugal filters. Then the necessary buffer solutions should be added. e) Both the samples (X and Y) should next be subjected to two -dimensional electrophoresis under the similar conditions so as to spot each sample protein on the electrophoresis gel “X” and “Y” respectively. e) Comparing the protein spots on both the sample gels X and Y. f) Identification of the protein spot which has migrated in the electrophoresis gel Y that has migrated through isoelectric focusing compared to the corresponding spot on the gel X as the identified phosphoprotein of interest (Leggat et al, 1995). The preservation buffer should contain a surfactant along with a transitional metal salt. There can be limitations with this method – this is because the concentration of sample protein used in the ordinary proteome analysis is very high, and hence precipitation of the proteins will tend to occur thereby decreasing the efficacy of phosphatase activity (Leggat et al, 1995). Solubilizing the protein by using a surfactant like SDS (sodium dodecyl sulfate) with addition of preservation buffers, the precipitation of the protein can be prevented and dephosphorylation may be performed without deactivating the phosphatase. However, high concentrations of such surfactants may deactivate the phosphatase and hence the transition metal salts are used to stabilize the phosphatase. The choice of preservation buffers are thus the alkyl sulfates like SDS and also alky sulfonates would be the surfactants of choice and the polyvalent transition metals belonging to group III to group VIII in the periodic table like manganese, tin, vanadium, iron or nickel are preferred as the transition metal salt. As a part of the buffer system EDTA salt, polyether compound, polyester compound and thio-ether component may be incorporated. Further the specific structure of phosphoproteins may also be determined and identified by comparing the results of the two-dimensional electrophoresis performed to the two- dimensional electrophoresis gel proteome databases (Leggat et al, 1995). Q2. Protein- Protein interactions to detect Signaling Pathways Protein- Protein interactions are attempts to identify the physical interactions between pairs or group of proteins. The study of these interactions elucidates signaling pathways and helps to model protein complexes for solving biochemical processes. The physical interactions may be inferred from a variety of experimental techniques like yeast two hybrid systems, affinity purification/mass spectrometry, from protein microarrays and fluorescence resonance energy transfer (FRET). The two distinct experimental approaches that will be used to study the protein -protein interactions involved in the signaling pathway in the given situation are selected as yeast two hybrid systems and affinity purification/mass spectrometry techniques ( Fromont et al,1997). i. The yeast two-hybrid system is a molecular biology based system which utilizes the principle that many eukaryotic transcription factors have discrete and uniquely separate DNA binding and transcription activity domains. So in this system one protein is fused to the DNA binding domain of the yeast GAL4 transcription factor while the second protein is fused to the GAL4 activation domain, because the GAL4 transcription factor has separate sites for DNA binding and transcriptional activation. Plasmids which encodes the two-hybrid proteins, one consisting of GAL4 DNA binding domain fused to protein P( protein of interest, referred as bait) and other consisting of GAL4 activation domain fused to protein Q( referred as prey) are transfected into yeast. If the proteins P and Q interact it leads to transcriptional activation of a reporter gene consisting of a binding site for GAL4. The interaction is estimated by assaying for the expression of a responsive reporter gene (Koegl and Uetz, 2008). Though the above method is powerful but it is subjected to several limitations. It cannot detect the interactions of more than two proteins or those depending upon post translational modifications. It is also incapable to detect variations in the state of protein- protein interactions under various cellular conditions (for example normal versus apoptotic cells).It cannot be applied to studies of kinetics of protein-protein interactions. The method can produce a large number of false positives statistically which means the interactions that are not of biological origin. One of the reason for false positives are the detected interactions may be unrelated to physiological processes. False negative results can also creep up for proteins not native to the nucleus. The two hybrid system interactions occur in the nucleus and many proteins are not in their native compartments. i. Mass spectrometry method consists of selective purification and enrichment of the bait protein with its interactors from a cell lysate. A protease enzyme such as trypsin is used to digest the isolated proteins into fragments of peptides. Mass spectrometry is used to analyze the protein mixtures and protein interactors are estimated from database searching. Immuno-precipitation or purifications are utilized to isolate the protein complexes. The method utilizes the tagged target proteins. Different variety of expression vectors with various tag sequences are designed for fusion to any target protein which can be cloned and expressed in a microbial host. cDNA clone coding for the tagged bait protein is engineered and cells are transfected with the clone and both expressed proteins and its interacting protein is purified by using the tag present on the bait. The properties of additional tag produce a handle for purification of fusion protein by affinity chromatography. These tags can range from one to few amino acids to complete proteins which can be attached to N or C terminal of the desired protein.GST and FLAG are the two common affinity tags. They provide one step purification process and have minimal effect on tertiary structure and hence on the biological activity of such interaction (Koegl and Uetz, 2008). ii. The major limitation of the above method is that the tagged protein might become over-expressed exaggerating the signal response, because it’s stoichiometry might not be similar to its partners in the complex and hence it must compete for complex assembly with non-tagged version of protein encoded in the genome. Often single step purification process is insufficient for recovering low abundance complexes from the background of over-expressed proteins. Q3. Detecting Nature of PI1P The principle of Fluorescence Resonance Energy Transfer (FRET) may be used to study the nature of binding. It is a phenomenon which occurs between two different chromophores (donor and acceptor) which have overlapping emission spectra separated by suitable orientation with a distance range of 10-80 angstroms. Intermolecular and intra-molecular FRET occurring between two spectrally green overlapping spectral protein variants fused to two different host proteins helps to provide monitoring of real-time protein- protein interactions or the conformational changes in the proteins. The intermolecular FRET may occur between one molecule (protein X) which is fused to the donor and other protein Y fused to acceptor. When the two proteins bind to each other FRET will produce a characteristic shift in emission spectra and when they dissociate the FRET will decrease. Using Fluorescence Digital Imaging microscopy it is possible to view location of GFPs in a living cell and follow the time course of the changes in FRET corresponding to cellular events at a very high resolution. This provides observation of dynamic molecular events in vivo which provides knowledge of significant binding kinetics ( Socher and Imperiali, 2013). With regard to present problem PI1P seems to be a pro-apoptotic factor and may be involved in inhibiting the tyrosine kinase activity (or by acting as a protein phosphatase) of the Growth Hormone Receptor by binding irreversibly with it. The logic is growth hormone will signal tyrosine kinase activity upon binding to its receptor on the cell membrane by becoming self phosphorylated and then phosphorylating the BAD protein, so it cannot attach to BclX-2. This will not lead to activation of Bax channels and calcium ions will not enter the mitochondria to eject the cytochrome C molecules which will not form the apaptosome complex. Hence there will be no formation of caspase 9 from procaspase 9. The caspase 9 will not be able to convert procaspase 3 to caspase 3. Then the caspase associated domain will be not be activated and endonuclease will not be stimulated to destroy the DNA and cause apoptosis, so the cell will grow as in tumor genesis. In case of P1IP, it prevents the kinase activity so BAD is activated and leads to apoptosis and hence this protein is anti-tumorigenic. It can be considered owing to the presence of phosphorylation sites it competes with BAD for the growth hormone receptor so the apoptotic protein BAD is not phosphorylated and inactivated and it causes apoptosis in spite of growth hormone –receptor interactions occur. In metastatic tumors it is down-regulated indicates it cannot compete with BAD and hence BAD is inactivated by phosphorylation and hence no apoptosis but metastasis takes place. This binding conformation of the P1IP and its competitor with the Growth hormone receptor can be detected by FRET. BAD will have a binding emission with the GH receptor and so the PIIP which can be detected. When the P1IP will dissociate from GH receptor emission will decrease and when associated emission will increase proving the binding kinetics. Q4. Appraisal of the High- Throughput Techniques to Identify and classify Tumor Type. Tumors or other lesions are group of heterogeneous population of cells that harbors the normal stromal and inflammatory cells in addition to the malignant cells. The presence of these cells would mask the transcriptional and genetic alterations that occur in the tumor cells. Hence cell isolation techniques are developed to identify the malignant cells from the non malignant cells, Often the amount of tissue extracted from the cancerous lesions are quite small that would be otherwise required for genome wide analysis (Eltoum, Siegal and Frost, 2002). These problems can be overcome by use of various amplification techniques like the polymerase chain reaction which uses degenerative oligonucleotide primers which permits relatively uniform amplification entire genome. Often RNA may be linearly amplified with the help of a polymerase for the global gene expression studies. In the past few years the specific role of cellular microenvironment including pro-apoptotic and anti-apoptotic proteins and signaling pathways is an active area of cancer research. The altered expression of p53 and PTEN ( phosphate and tensin homolog) have been extensively studied in neoplastic epithelium and stroma. Serial Analysis of Gene Expression as a High throughput is used to determine the extensive transcriptional changes that occur in these proteins in various in all types of cancer cells during the progression of cancer, but the genomic alterations are only detected in the epithelial cancer cells. Therefore the molecular characterization of every cell type will elucidate our understanding about the role of these epithelial cells in tumorigenesis and provide molecular targets for cancer intervention and treatment. Genome wide molecular profiling applications or the analysis of gene expression profile may provide insights to the changes in the biochemical and molecular pathways that occurs when there is a malignant transformation and indicates the direction of cancer progression. The transcriptional profiles corresponding to thousands of genes can be established by global profiling techniques like the SAGE and the tissue microarray analysis (Rennstam and Hedenfalk, 2006). The microanalysis method is based on hybridization of cDNA samples to immobilized probes on the microarray analysis slides. Formalin fixed, paraffin wax embedded tissue for multiple tumor tissue microarray construction are normally being used. The analysis is based on finding the stain characters (diamino-benzidine tetrahydrochloride is used as chromogen stain) of the positive results with the negative controls (Rennstam and Hedenfalk, 2006). . The SAGE method on the other hand produces a library of expressed genes by considering the raw count of the sequence tags each of which represents a transcript in the RNA population. Since the method can quantify the expressed genes precisely it permits the creation of various gene expression profiles which may be compared with other cell or tissue types. The major usefulness of the SAGE methodology is in the concept that there is no requirement of prior knowledge base of the sequences to be analyzed. The microarray analysis can be applied to the analysis of large sample tests. The reproducibility of results with SAGE and Microarray analysis are very well correlated (Ishi et al, 2000). The array based CGH (Comparative Genomic Hybridization) approach may be used to identify the high resolution global genomic changes acquired during the progression of cancer. In this array based CGH, the differentially test DNA (from the tumor cells) is co-hybridized with a normal DNA onto a representation on the genome, which constitutes multitude printed spots of the target DNA. Microarrays made from the cDNA are mostly used for this purpose. However the use of DNA clones as the target of the genomic DNAs are hampered by the suboptimal hybridization of the intron specific genetic material, present in the genomic DNA but which is absent in the cDNA (Rennstam and Hedenfalk, 2006). Bacterial Artificial Chromosome Arrays (BAC arrays), utilizes the segments of Human Genomic DNA as the targets of hybridization and these BAC arrays provide a average resolution of around 80 kilobases (Rennstam and Hedenfalk, 2006). The High density oligonucleotide arrays although have a greater resolution than the BAC arrasys but are usually non tiling in nature (Rennstam and Hedenfalk, 2006). The custom made arrays provides the advantage of individual probe design of the order of single exon resolution. Therefore by the comparison of differential gene expressions profiles which are established by cDNA microarrays between the normal cells, preliminary invasive carcinoma and the metastatic cells, it is possible to detect the genes which are directly associated with each tumor stage in the tumor development process. This indicates the comprehensive identification metastasis related genes present in the clinical biopsies (Abba et al, 2004). On the other hand combination of laser capture micro-dissection and DNA microarrays which generates gene expression profiles of pre-maligmant , pre-invasive and invasive stages of cancer tissues it is possible to conclude various and distinct stages of progression suggesting the transcriptional alterations elucidating the potential growth which are present in the pre-invasive stages (Mimori et al, 2005). References Abba MC, Drake JA, Hawkins KA, Hu Y, Sun H, Notcovich C, Gaddis S, Sahin A, Baggerly K, Aldaz CM (2004) Transcriptomic changes in human breast cancer progression as determined by serial analysis of gene expression. Breast Cancer Res, 6:R499-R513. Eltoum IA, Siegal GP, Frost AR(2002): Microdissection of histologic sections: past, present, and future. Adv Anat Pathol , 9: 316-322. Elke Socher and Barbara Imperiali.(2013) FRET-Capture: A Sensitive Method for the Detection of Dynamic Protein Interactions. ChemBioChem, Vol 14,Issue 1, 53-57 Fromont-Racine, Micheline; Rain, Jean-Christophe, Legrain, Pierre (1997). "Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens". Nature Genetics 16 (3): 277–282 Ishii M, Hashimoto S, Tsutsumi S, Wada Y, Matsushima K, Kodama T, Aburatani H (2000): Direct comparison of GeneChip and SAGE on the quantitative accuracy in transcript Profiling analysis. Genomics 2000, 68:136-143. Karin Rennstam and Ingrid Hedenfalk (2006). High-throughput genomic technology in research and clinical management of breast cancer :Molecular signatures of progression from benign epithelium to metastatic breast cancer Breast Cancer Research 2006, 8:213 Koegl, M.; Uetz, P. (2008). "Improving yeast two-hybrid screening systems". Briefings in Functional Genomics and Proteomics 6 (4): 302–312 Mimori K, Kataoka A, Yoshinaga K, Ohta M, Sagara Y, Yoshikawa Y, Ohno S, Barnard GF, Mori M (2005): Identification of molecular markers for metastasis-related genes in primary breast cancer cells. Clin Exp Metastasis, 22:59-67. M. Butler et al.(1986), Journal of Neurochemistry, vol. 47, No. 5, pp. 1520, 1521. Robert W. Leggett et al.(1995), The Journal of Biological Chemistry, vol. 270, No. 43, p. 25882. . Read More