Extract-iTRAQ-Labels

  1. Create a directory c:\MascotCommandLineTool\
  2. Copy perl Scripts to this directory. You will have:
    1. C:\MascotCommandLineTool\MascotqueryParse.pl
    2. C:\MascotCommandLineTool\ProteinCalculate.pl
  3. Confirm you have perl installed on your computer (and check the version-I'm using v5.8.8) by executing the command:
    • perl -v
  4. Execute (Run) the Perl Script MascotqueryParse.pl including the iTRAQ Labels as arguments. For example:
    • Run MascotQuery.pl perl script on Mascot Results with this command.
    • From the Start Menu-> Run...
    • In the Open field type: 'cmd ' for the command line window
    • type: cd c:\MascotCommandLineTool\
    • with the prompt c:\MascotCommandLIneTool\
    • type: MascotQueryParse.pl F014587.csv 114 116
    • The Exported Mascot Result file (F014587) is processed, extracted the iTRAQ values and using the ratio (114/116)
  5. In the Task manager watch the processor chart showing processor activity as the program executes-
    1. parsing the file, and processing the query files, extracting the iTRAQ values from all of the Queries, then reassembling them to the queries used in the protein IDs
  6. Two output files are created: F014587.csv.out, F014587.csv.protein.out
  7. These files should be about 10% the size of the orginal file, usually 2-5 Mb in size. They are not too large for excel. Open F014587.csv.out and also open Normalization.xls
  8. Copy the worksheet from F014587.csv.out to Normalization.xls by right-clicking on the tab for the worksheet, clicking the box for copy and selecting Normalization from the drop-down window.
  9. In Normalization activate the new worksheet select all and copy to the clipboard, then activate the 'Complete QueryData' tab in the Normalization Workbook and from the Edit menu select 'Paste Special...' and then paste as values. This eliminates any formulas and is the safest way to ensure data is transferred and you don't need to worry about problematic formulas referencing missing data.
  10. Run the Macro 'Generate Peptide Stats' -This macro takes as input a list of all the queries from a Mascot Search Result,
    1. It inserts a column for ratios of 117/115
    2. It sorts then filters the 115 iTRAQ values to remove the queries that don't have a minimum intensity of 15
    3. It sorts then filters the 117 iTRAQ values to remove the queries that don't have a minimum intensity of 15
    4. It inserts Extra Rows at the top to calculate summary statistics (Median, Mean, Total Count) for each iTRAQ tag 114, 115, 116, 117 and the ratio(117/115)
    5. It is not complicated, but the macro must be editted and revised when different iTRAQ labels are used (for example 116/114 or 115/117)
  11. Select the Tab 'Peptides Stats' and write down the Median Ratio in Cell 'L2' - this is used as the Peptide Normalization Factor in the second PerlScript
    1. Pappin et Al from ABI describe this factor to adjust the iTRAQ ratio and normalize it. Since equal amounts of material were initially labelled with each iTRAQ tag (ie 50 ug of AMD Bruch's Membrane and 50 ug of Normal Bruch's membrane) we need to normalize our peptide ratios and set the median ratio of all observed masses to 1.0. If the median is 1.12, then all observed masses are too large and in every case they can be divided by 1.12 thereby reporting the 'peptide-normalized' ratio.
  12. From the Command line execute:
  13. c:\MascotCommandLIneTool\ProteinCalculate.pl F014587.csv.Protein.out 1.12
  14. The output files from this command include:
    1. F014587.csv.Proteins.csv.SummedProteinValues.out (2-5 Mb),
    2. F014587.csv.Proteins.csv.ConciseListOneHitWonder.out (<10Kb)
    3. F014587.csv.Proteins.csv.ConciseListAll.out (<10Kb)
    4. F014587.csv.Proteins.csv.ConciseList2orMorePeptides.out (<10Kb)
  15. F014587.csv.Proteins.csv.SummedProteinValues.out now has iTRAQ ratios calculated for each protein identified in the search results. The format is comprehensive, expanded and detailed. Open the file in Excel and copy the worksheet into an Excel workbook, rename the sheet by stripping the name to only the Mascot Search Index number (F014587(Detailed))- Save the Result Excel workbook.