Protein Databases Lysis and Protein

Question: 1 .Briefly describe how this gel was created. 2. Name 3 ways you could identify the protein located at particular spot on the gel? Which method would you use if the proteins were from a human sample, and why? Which method would you use if the proteins were from a bacteria with an unknown DNA sequence, and why? 3. How many proteins are located on the X and the Y chromosomes? 4. Under Subcellular location, based on direct assays only, in which two cellular components can this protein be found?5. Name the three PTMs that have been associated with human disorders 6. Briefly describe two strategies for determination of phosphorylation sites using mass spectrometry (MS) 7. In addition to MS, what other method was used to characterise this site? 8. Which tissue has the highest level of expression for this protein? Which tissue has the lowest? 9. Name one tissue where the mRNA levels are low, but the protein levels are high.10.How might this be explained? 11.What does this tell you about the information content of transcriptomics analyses?12. Do you think the interaction with SOD1 is real, why or why not? 13. Do you think the interaction with ATXN2 is real, why or why not? 14. What is the position of the two RRM domains?15. What does RRM stand for and what is the function of this domain?16. How many other human proteins have RRM domains? Answer: Two dimensional polyacrylamide gel electrophoresis is known as 2D-PAGE. A 2D gel is created by following ways: Lysis and Protein Extraction from cells This preparation method provides total cellular protein samples that are free of contaminating nucleic acids and free of protease activity. The protein extracts are used for analysis of proteins by two-dimensional gel electrophoresis. Determining Protein Concentration of cell lysates Perform a Bradford protein assay to determine protein concentration. First dimension Separation: Isoelectric Focusing Second dimension Separation: SDS-Page Transfer of iso-electric focused gels to SDS-PAGE gels Detecting proteins and staining the gels The three processes for the identification are : Edman degradation Amino acid analysis Peptide mass fingerprintng The method which can be used for a human sample is peptide mass fingerprinting as this method relied on preexisting data which already exist for human. Whereas for bacterial sample one has to go about with Edman degradation with an unknown DNA sequence. The total number of proteins made are 70,611 Number of genes in chromosome X = 1927 Number of genes in chromosome Y=89 The two cellular locations are: Nucleus and nucleoplasm. The post translational modifications which are commonly linked to human disease are : Protein carbonylation in Alcoholic Liver Disease. Protein hydroxylation in autoimmune polyendocrine syndrome (APS) type I Protein hyper-acetylation in Alzhemiers Disease. Strategies for determination of phosphorylation sites in proteins. Identification of phosphopeptides by peptide mass fingerprinting. In this method, phosphopeptides are identified by comparing the mass spectrum of an untreated sample to that of a sample treated with phosphatase. In the phosphatase-treated sample, potential phosphopeptides are identified by a decrease in mass due to loss of a phosphate group (80 Da). Phosphorylation sites can be identified by peptide sequencing using MS/MS. Edman degradation can be used to monitor the release of inorganic 32P to provide information about phosphorylation sites in peptides. The highest protein expression is in salivary glands and lowest in smooth muscles The mRNA level is high and protein level is low in pancreas. The pancreas is a composite organ with both exocrine and endocrine functions hence it is expected that the protein level be high compared to the mRNA level. This shows that the RNA coding for the nuclear RNA/DNA-binding protein that functions in RNA processing and metabolism, including RNA transcription, splicing, transport, and stability has a longer half-life. The interaction between TARDBP and SOD1 is not real as there is no experimental evidence. The interaction between ATXN2 and TARDBP is however real as there are both experimental and text-mining evidence to support this. The two RRM domains. RRM 1 spans from 104 to 200 residues while RRM2 spans from 191 to 262. RRM is actually RNA Recognition Domain which is responsible for binding RNA and DNA. There are 263 Human proteins with RRM domain among which 235 are reviewed and 28 are un-reviewed.