Thursday, February 20, 2014

N15 labeling in Proteome Discoverer

This question pops up a lot, despite the fact that this workflow is available on both the BRIMS portal and PlanetOrbitrap.  It never hurts to put it up in an additional place, right?

Question:  Can I do N15 labeling quan in Proteome Discoverer
Answer:  Yup!  But its a little tricky.

This link will take you to a Powerpoint with some instructions and an XML file that has a method I made for someone for PD 1.3.

Ultimately, the real trick is adding the modifications you need for each amino acid.  You need specify these mods uniquely by how many nitrogens are in each of your amino acids.

First start by updating your Unimod database.

Then add these labels.

Once these are in there, you can basically build the method based any of the precursor based quan methods (like SILAC). Comparing your light to your heavy.

Now, it gets a whole lot trickier if you are looking for incomplete incorporation.  You begin to run outside the realm of what you can do with the SILAC node.  What you need to do at that point is run your N15 mods as dynamic and use the precursor ion area detector and some database work will be necessary.  It certainly isn't impossible, but it will make for a nice long day of analysis.

The XML method in this file assumes that you are trying to compare two things, channel 1 with no N15 and channel two with 100% incorporation of N15 into each amino acid.  If you alkylate your cysteines you will need to make a custom modification that includes the mass shift of your N15 on cysteine as well as the mass shift of your alkylation (cause we can't yet do 2 mods/amino acid).

EDIT: Here is the new download link for the PD 1.3 slide deck.


  1. Hi Ben, nice blog! And I'm a fan of PD too!
    Recently I'm trying 15N labelling to perform quantification in my experiment, this is exactly what I'm looking for.
    The attachments of .pptx seem broken now, could you please upload a .pdf version? Thank you in advance!

  2. This comment has been removed by the author.

  3. Hi Ben,
    Thank you for this useful information.
    Now I'm doing plant N15 labeling phosphoproteomics analysis, and I'd like to use PD to search N15 phosphopeptide results. However, Sequest can't allow two mods/amino aicd as you mentioned for N15-labeled Ser/Thr/Tyr, so I'd like to know if you have any suggestions for using PD searching. Thank you so much.

  4. Yeah, thats tricky. If it was all static I'd search with a "super modification" that has the mass of the phospho+the mass of the N15 incorporation, would it work with a dynamic modification? I'm not sure....