SILAC / AQUA

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When using SILAC and AQUA, you are using synthetic amino acid residues containing heavy isotopes, typically 13C and 15N, but may also use deuterium. How can you define these in GPMAW?

For a start you need to open the ‘Edit mass file dialog’, by going to the main menu and select Edit | Edit mass file. You then click on the ‘Atomic weights tab and scroll to the bottom of the list. Here you replace some of the entries if you have all entries defined, if you have less, you add to the list.

In the first column you enter a 1 or 2 letter abbreviation of the atom, Default for standard mass file is C3 for Carbon13, D for Deuterium and N5 for Nitrogen15. Alternatively you can call them Cx and Nx. As you are working with isotopes, the average mass does not make sense, so you just enter the same value for  average and monoisotopic mass as outlined in the figure below.

Atomic weights

You then click OK and close window. The values in the atom table is saved in the .ini file, not in the residue mass file, this means that you have to close GPMAW and reopen it to load the mass values.

You then restart GPMAW. From the main menu, you select the relevant residue mass file you want to work with (e.g. if you want to work with standard reduced/oxidized Cys you select aa_mass, if you work with iodoacetamide derivatized Cys you select acetamid). You then open the Edit mass file dialog on the Mass file tab.

EditSILAC

To edit lysine, click on the composition of Lys and select the small calculator button to open the elemental composition editor. Setting the SILAC 13C6 lysine residue, you set Carbon to 0 (zero) and the newly created Carbon13 C3/Cx to 6. Select OK and edit any other residues that need to be changed (e.g. arginine for the typical SILAC experiment).

You then select the Save as button and save the the table as a new residue mass file (e.g. with the name SILAC). Close the dialog, close GPMAW and restart the program, and the new mass file will be available for selection in the drop-down box in the main toolbar.

Working with both heavy and normal residues:

If you need to work with normal and heavy residues in the same sequence, you need to define the heavy residues as new residues, using the positions 21 to 30 in the residue mass table. Remember that each residue needs a unique 1-letter code to function. Save the file under a new name.
You then have to edit your sequence to put the heavy residues into place.

If you ‘just’ want to compare the mass values of normal and heavy residues, you can do that in the peptide list (from a digest) by using the secondary mass table feature of the peptide window.

Site last updated: October 03, 2017

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