5:010110MEllmOel;1 52%. Instructions for proteomics data analysis.pdf – Say.4 1Instructions for proteomics data analysis Background: Scribble is a scaffolding protein involved in the regulation of cell polarity. It is a tumour suppressor and is frequently deregulated in cancer. In this study we have expressed human Scribble as GFP-tagged fusion protein in human embryonic kidney cells (HEK293). In a parallel control experiment we expressed GFP alone in the same type of cells. The two cell cultures were harvested, the total cellular proteins were extracted and GFP and GFP-Scribble were immunoprecipitated with a monoclonal anti-GFP antibody. The immunoprecipitated proteins were digested with trypsin and analysed by LC-MS/MS. The provided mgf files contain the results from this analysis.Your task is to: Identify proteins that specifically associate with Scribble. These were the ones which are found in the sample immunoprecipitated from GFP-Scribble-expressing cells but not found in the sample obtained from cells expressing only GFP. Important: Scribble is the bait proteins. Do not list it among the “Scribble-interacting proteins… This is a very common mistake students have made in previous years.Study the literature to find out what is known about the functions of the identified Scribble-binding proteins. 3. Write a report that present the results from 1 and 2. Approach:Essentially you need to follow the same approach we followed in practical 6. You will use Mascot to process the two files, then export the results to Excel, consolidate the two table, perform the G-test and Bonferroni correction and then select the candidate proteins based on the Bonferroni-adjusted p-values. in the report you can illustrate the results with a table and also with selected MS/MS spectra from the Mascot output.Report: Write it as a short SPF paper with title, abstract, short introduction, methods, results and discussion, and references.Title: should be short and informative. Should indicate what the most important result was. Avoid abbreviations in the title.Abstract: Should be no more than 150 words and should give enough Information about the aims, methods used and results obtained. Avoid technicalities such as volumes of reagents, cell growth conditions and statistical test used. This should be described in the methods section.Introduction: brief description of Scribble with 4-5 references cited. Brief introduction into proteomics and how it is used to study protein-protein interactions. At the end of the introduction write a paragraph to define the main aim and specific objectives. The introduction should be no more than 200-250 words.Methods: very briefly describe the Mascot search and statistical tests used. Refer to Metodieva et al (2016) for the cell transfection, growth and immunoprecipitation. No more than 150-200 words for the methods.Results and discussion: the remaining 300-400 words should be used to present the results and discuss them. You can merge results and discussion into one section. The results should have tables and perhaps a figure or two from the Mascot search results. It could be a MS/MS spectrum with assignments of y and b fragments for one of the identified interacting proteins.Start with the results section with a text paragraph, not a table or figure. It could be a summary of the results. Then present the results shown in tables and figures. Then discuss and complete with a short paragraph of conclusions.References: cite 10-12 references at most. Cite only papers from serious peer-reviewed journals, do not use websites as sources of information. Use Harvard style throughout. Use Endnote if possible. Each reference in the list should be cited at least once in the text and each citation in the text should be present as a reference in the list.
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