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Change the Precursor ion tolerance setting to 25 ppm.Choose TDP43DB.fasta as the Protein Database and TDP43_fixed.mgf as the input peak list.Select the Search GUI tool from the MS MS Search category in the tool menu.This means that all of the heavy lifting is done by high performance computers that are well suited to the task. In this tutorial we will instead use Galaxy to run SearchGUI, which in turn will run Tandem MS searches using many search engines on a server. Tandem MS database search on large datasets is a task that can reduce laptops and most desktops to smoke and ruin. The SearchGUI program itself can be run directly on your desktop or laptop computer, but for anything other than small datasets this is not advisable. Rename the resulting output file in your history to TDP43_fixed.mgf.Choose TDP43.mgf as the input file and run the tool.Select the Fix Bruker MGF tool from the Other Proteomics tools menu in Galaxy.The solution is to run the data through a tool called Fix Bruker MGF in Galaxy. Bruker like to embed non-standard information into their mgf files but this confuses all non-Bruker software. The TDP43.mgf data file is an mgf formatted file produced by the microTOF-Q, which is a Bruker instrument. The tool will accept multiple sequences in the paste box provided they are in FASTA format. Note that if you have several in-house sequences or you suspect that your protein might have an alternative sequence you should include all those in the database. Rename the database in your history from Pasted Sequences to TDP43DB.fasta.
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Run the tool and a new entry should appear in your history with the TDP43 sequence as well as a selection of contaminant sequences to act as a control.Select the option to modify identifiers for compatibility with SearchGUI/PeptideShaker.Check the box the append known cRAP sequences.Paste the entire fasta entry for TDP43 (including the identifier line and all sequence lines) into the required box.Select the Paste Sequences tool under Fasta Manipulation in the tool menu in Galaxy.To create a protein database containing this sequence GNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGM GGFGNQGGFGNSRGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPS TGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREFįSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNP LVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLK MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGN The amino acid sequence of our protein is given below in FASTA format >TDP43 Import tutorial data into the newly created history.Create a new history called "Peptide Shaker Tutorial".Step 1: Create a new history and import data This is a large file so if you haven't done it yet you should start downloading before you proceed.
#Peptideshaker download download
Important Note: In order to perform the later steps in this tutorial you will need to download peptide shaker. This tutorial covers all the tools required in this situation. It is often the case in such experiments that the protein of interest has an amino-acid sequence that is not yet published, so searching standard databases such as Uniprot is not an option. You would like to cut out the band of interest and use Tandem Mass Spec to confirm that its amino acid sequence corresponds to what you expect. In order to illustrate the use of this tool, imagine a scenario where you have run a gel and suspect that a particular band corresponds to a specific protein of interest. These tools essentially automate the many steps illustrated in the Multiple Search Engine Tutorial. This tutorial introduces two very useful tools SearchGUI and PeptideShaker that allow a comprehensive multi engine search and downstream statistical analyses to be performed in just two steps. Use PeptideShaker and SearchGUI to search a custom protein database