Phosphorylation Variable modification

Phosphorylation Variable modification dawn dufield  2020-04-13

I am looking for some phosphorylated peptides and was wondering how your variable modification worked? I added 2 sites of phosphorylation as variable in a specific peptide and only see it predicting the double phosphorylation as a potential MRM. I was hoping it would give me none, 1 (either on 1 site or other) and both.

Any help would be appreciated


Nick Shulman responded:  2020-04-13
This is the webinar that we always point people at when they have questions about variable and explicit modifications:

If you want to modify one peptide in a particular way, it is probably easiest to right-click on it in the Targets tree and choose "Modify". This allows you to choose from any of the modifications that you have listed at "Settings > Peptide Settings > Modifications".

If you want to be the one that decides which modifications are applied to which peptide, then you probably want the modification to be "Explicit", which means that it appears in the list at "Settings > Peptide Settings > Modifications", but the checkbox next to the modification in that list is unchecked.

If Skyline is not giving you the permutations of variable modifications that you want, there are a number of different things that might be going wrong. It might be easiest if you sent us your Skyline document. (you can "File > Share" to create a .zip file).

Hope this helps,
-- Nick
dawn dufield responded:  2020-04-13
Hi Nick,

I know how you can modify a specific residue within the tree and put on a modification on that residue. I guess I'm asking the question on whether skyline can predict the various MRM's for a peptide that might have a couple possibilities without explicitly modifying and making each one.

Lets say this is your peptide

GSPDIVSLR. Can you just enter this with a variable modification on Ser to be +/- phosphorylation? so it would have 4 choices, none, 1 phospho at first serine, 1 phospho at 2nd serine or 2 phos at both serine?

I know I can make 4 copies of this peptide and select each and both or none, but was hoping there was an automatic way it would do that for me if I said variable modification on Serine residues?

Hope that makes sense
Nick Shulman responded:  2020-04-13
Yes, things are supposed to behave the way that you're describing.

There are a few things that might cause this to not be happening.

One is that Skyline only automatically adds the permutations of peptide modifications if it's a real Protein (such as something you got by importing a FASTA file). If it's just a list of peptides that you got by doing "Edit > Insert > Peptides", then Skyline will not add or remove modification variants for the peptides that are there.

The other thing to check is to make sure that "Auto Select Peptides" is enabled for the Protein. One way to do that is to right-click on the Protein in the Targets tree, and choose "Pick Children" and make sure that the magic wand icon is pressed. (You can also use the Document Grid to change Auto Select Peptides).

I am not sure, but there also might be some interaction of this with the settings that you have at "Settings > Peptide Settings > Library".

If these do not fix it for you, you should send us your Skyline document.
-- Nick
dawn dufield responded:  2020-04-13
Thanks Nick,

Looks like it was not imported as a fasta, but pasted in as a protein sequence. I got it to work if I reimported as fasta.txt.

One more question. If there is only a couple peptides that I need to have it do all the modifications, do I need to delete all the rest? or what is the easiest way to filter?

dawn dufield responded:  2020-04-13
Is there also just Ser modification not a Thr? or do I need to make that one as a custom?