Custom digest site

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These are the steps I took to add a new enzyme cleavage site to the TPP. I had to do this in order to run TPP on a data set that used an non-standard enzyme.


Preface

I did this on the linux version of TPP. I have no idea if this will work on the Windows distribution. These instructions assume you know how to compile TPP on linux. They also assume you are comfortable editing C code. Both are a pre-requisites for these instructions.


Warning

These instructions worked for me, I hope they work for you but don't blame me if they screw up your installation. BACK UP YOUR DATA FIRST!!


Steps

  • Download the latest version of TPP from SourceForge. As of this writing I used version 4.3.1.
  • Unpack the zip archive.
  • Move to the src directory and open the file src/Enzyme/ProteolyticEnzymeFactory/ProteolyticEnzymeFactory.cxx using your favourite file editor.
  • Follow the syntax provided for the struct called const char *ProteolyticEnzymeFactory::CATALOG_ENZYMES[][4] and add your enzyme information there.
For instance, the template for trypsin is:
 {"trypsin","true",NULL,"KR|P|false"}
Your custom enzyme might look like this:
 {"dingdong", "true", NULL, "TYRK|H|false"}


  • Next, edit the function called void ProteolyticEnzymeFactory::showEnzymesCatalog() following the syntax provided there too.
For 'stricttrypsin' you see:
 ,"stricttrypsin"
Insert the name of your custom enzyme after it:
 ,"dingdong"


  • Now edit the function called ProteolyticEnzyme* ProteolyticEnzymeFactory::getProteolyticEnzyme(const char* input_name) following its syntax as a template.
For 'stricttrypsin' you see:
   else if(strcmp(name, "stricttrypsin") == 0) {
   enz = new ProteolyticEnzyme(name, fidelity, True, NULL);
   enz->enterSpecificity("KR", "", False, min_spacing_);
   enz->fixSpecificity();
 }
Copy this syntax exactly and replace where needed to match your enzyme
 else if(strcmp(name, "dinddong") == 0) {
   enz = new ProteolyticEnzyme(name, fidelity, True, NULL);
   enz->enterSpecificity("TYRK", "H", False, min_spacing_);
   enz->fixSpecificity();
 }
  • Save this file and close it.


  • Next, open src/Visualization/xinteract/xinteract.cxx for editing
  • For the function called const char* getSampleEnzyme(char e) add an entry to represent your enzyme. Pick a single character (letter or number) not already allocated to some other enzyme.
For 'trypsin' you see:
 case 'T': return "trypsin";
Add your enzyme right below it:
 case 'Y': return "dingdong";
  • In the int main function you'll find an if-conditional (if(argc < 2)). You need to add the enzyme digest "flag" for your custom enzyme to the the cout command for all the other enzymes.
Look for this line:
 cout << "                     -eS [specify sample enzyme = StrictTrypsin]" << endl;
Again, copying it's syntax we add this line right below it:
 cout << "                     -eY [specify sample enzyme = dingdong]" << endl;
  • Save the file and close it.


  • You need to edit the programs that parse RAW search engine results (i.e.: Tandem2XML, Mascot2XML, etc...) to recognize your enzyme pattern. These instructions only cover X!Tandem since that is the search engine I use.
  • Open src/Parsers/Algorithm2XML/Tandem2XML/TandemParamsParser.cxx
  • Edit the function called void TandemParamsParser::initEnzymes() and following the syntax of other enzymes, add yours.
For 'chymotrypsin' you see:
 addEnzyme("chymotrypsin", "[FMWY]|{P}");
Add your enzyme right after it:
 addEnzyme("dingdong", "[TYRK]|{H}");
  • Save the file and close it


  • Now move back to the src directory and compile TPP as you normally would.




These instructions worked for me. Hopefully I haven't left anything out. In case I did please feel free to update the site.

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