CSB450-Assignment-2023-

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Jan 9, 2024

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University of Toronto Department of Cell and Systems Biology CSB450 Course Assignment Professor Dinesh Christendat This is not a group assignment. Please do not meet with your group members to discuss or answer these questions. Please note: These questions will take a significant amount of your time so get a head start early. No aids allowed – Do not use AI such as ChatGPT to answer these questions. DUE: Thursday November 16, 2023 at 5pm

2 1. The rhizosphere is the zone surrounding the root of a plant and involves a complex relationship between the microbial population, the soil, and the plant. Under certain environmental conditions, different organisms flourish because of symbiotic relationships with the plant, such as nitrogen-fixing microbes. ( 10 marks ) Assuming these organisms are all sequenced and show distinct genomic composition , design an experiment to determine which organisms would be the most abundant in the rhizosphere in the spring. Helpful guidelines : Use 2D gels for your analysis. Please provide a strong rationale for your choice of stain and the number of replicates you will be conducting. You can use specific proteins from each organism as biomarkers for your analysis. 2. ( 10 marks ) Quantitative proteomics has become a popular approach to understand how organisms respond to changing condition. There are two main approaches for quantitative analysis with LC-MS; in-vitro labeling and in-vivo metabolic labeling of proteins. a) Describe in detail three factors that need to be optimized for metabolic labeling of proteins in-vivo . Use diagrams to explain your answer. (5 marks) b) Can you think of advantages for using metabolic labeling vs in-vitro modification of proteins for quantitative analysis? (5 marks) 3. ( 15 marks ) a) Using the spectra above, determine the average molecular weight of this protein. Hint . Use at least 3 charged states in your calculation. Show all calculations. b) Using a well labeled figure describe the principles of protein separation by 2D gel electrophoresis. Compare and contrast detection limits and linear dynamic range of stains in 2D gels. Use the 2D gel diagram to illustrate your answer .

3 e) Describe the principles of two-dimensional peptide separation with a diagram . You should explain in detail the purpose for each separation column in this process. 4. ( 5 marks ) Use the protein sequence below to answer this question: WINDYRDDGQSTYGSKVSFTIEKMTGIVKWFNADKGFGFITPDDGRSDVFVHFS AIQNDGYKSLDE a) Generate a tryptic peptide pattern for the sequence above. b) Using the 2 nd peptide from your digestion, generate an MS-MS fragmentation pattern, showing the y- and b- ions and the masses for the ions. Just use a diagram to show the fragmentation, ions, and masses. 5. ( 20 marks ) a) The following questions pertain to protein complementation analysis. 1. With the aid of a diagram ( flowchart ), clearly illustrate the principles of PCA. Provide appropriate control (including positive control) to demonstrate that this approach can be applied to detect protein-protein interactions. You can use the DHFR system and explain. 2. What are the major advantages for using PCA over Y2H analysis? b) Please do not use PCA for this question . Describe an approach to obtain Arabidopsis thaliana membrane protein interactome map. Explain in detail, with the aid of a diagram, the principles of this application. In your answer, explain your reasoning for choosing this approach. Use a diagram and include all controls. 6. ( 30 marks ) The shikimate pathway enzyme DAHP synthase is highly regulated with the accumulation of Tyrosine in plants and microbes. The mechanism of regulation in plants is unique: Tyrosine accumulation leads to phosphorylation of DAHP2 and its interaction with a 14-3-3 protein prevents DAHP2 from entering the chloroplast where it is needed. While the phosphorylated-DAHP2-14-3-3 protein complex resides in the cytoplasm it becomes ubiquitinated and the whole complex is targeted for degradation by the 26S proteosome. However, it is thought that glycosylation of this ubiquitinated complex can rescue from turnover by the proteosome.

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4 Design experiments to investigate the following: 1) The protein complex phosphorylated-DAHP2-14-3-3 undergoes ubiquitination and this leads to its turnover. 2) Identify which protein in the complex becomes ubiquitinated 3) The protein complex phosphorylated-DAHP2-14-3-3 undergoes glycosylation. 4) Identify which protein in the complex undergoes glycosylation 5) Identification of the kinase responsible for the phosphorylation of DAHP2 6) Demonstrate that the kinase you identified can be inhibited with the use of a known inhibitor I. Hint: Use at least 4 different concentrations of inhibitor I and LC-MS with some type of labeling approach for quantitative analysis. Hint: You should use one or more diagrams to answer each part of this question. Note: you will obtain 10 additional marks if you obtain at least 25 marks for question 6.

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