Special SIL peptide standard library

support
Special SIL peptide standard library lyjgbb  2020-06-10
 

Hi Skyline Team,

I wish Skyline could have a function to allow us to build a special SIL peptide standard library, which includes the customized stable-isotope labeled peptides as standards for our target LC-MS quantification on the unlabeled peptides from either cells or tumor samples. Because we are focusing on the HLA peptides, which are not typical tryptic peptides but they have a kind of diverse peptide sequences, we have a very large SIL peptide library with peptides labeled on diverse amino acids. When we run different samples, we have to choose different SIL peptides from the library, input the corresponding light partners first, and then manually modify the amino acids into SIL version one by one. So this process is very time-consuming and has risks for many human errors. It will be greatly helpful if we can set up a SIL peptide library first in Skyline and then choose the selected SIL peptides per unique sample efficiently from the library, by just inserting the light partners into Skyline. That means Skyline will give users a chance to choose which library (typical protein database or SIL peptide library) they would like to match the peptides with when they insert peptide sequences into the program.

Thank you so much for your great efforts and kind consideration!
John

 
 
Nick Shulman responded:  2020-06-10
I am not sure I understand your question.

Can you send us your Skyline document?
In Skyline, you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including spectral libraries.

If that .zip file is less than 50MB you can attach it to this support request.
Otherwise, you can upload it here:
https://skyline.ms/files.url

Let us know which peptide sequences you are trying to insert. We will figure out whether there is something you could do at "Settings > Peptide Settings > Modifications" which would cause the correct variants of those peptides to be inserted, or if there is some other possible workaround.
-- Nick
 
lyjgbb responded:  2020-06-11
Hi Nick,

Thank you for your quick response!
Attached please find two Skyline files with different selected standards from our SIL peptide library as target lists for LC-PRM-MS quantification purpose.
As you may notice in the files, the SIL labeled AAs on these sequences are diversified and thus there is no way to set up a globe definition of amino acid modification. So as I mentioned in my request, the best way for us is that to build a SIL peptide library into Skyline, and then we can insert a unique list of the light peptides of interests into Skyline to have the corresponding SIL peptide standards matched and imported from the preexisting SIL library in Skyline. By doing this, we don't need manually change the AA modifications for each peptide after importing the light peptides as what we are doing now. A lots of human errors can be avoided and a lots of time can be saved.

Thanks!
John
 
Nick Shulman responded:  2020-06-11
It sounds like you have been using the "Modify Peptide" menu item, and you want a way to paste in a list of modified peptides.

When you do:
Edit > Insert > Peptides
in Skyline, you can specify modified sequences in the "Peptide Sequence" column.

So, you could paste the following into the Insert Peptides dialog:
MPMQDIKMIL[Label:13C(6)15N(1) (L)]
MPMQDI[Label:13C(6)15N(1) (I)]KM
TLYNPERTITV[Label:13C(5)15N(1) V]
NPEAIEDNKL[Label:13C(6)15N(1) (L)]
NYTTVPQEL[Label:13C(6)15N(1) (L)]
QENTQTPTV[Label:13C(5)15N(1) V]
SAIESTILL[Label:13C(6)15N(1) (L)]
NAYDFI[Label:13C(6)15N(1) (I)]MEF
MAIGETLVL[Label:13C(6)15N(1) (L)]
SEKVVQHAL[Label:13C(6)15N(1) (L)]
MPVSAFTVIL[Label:13C(6)15N(1) (L)]
MPVSAFTVI[Label:13C(6)15N(1) (I)]
SEYV[Label:13C(5)15N(1) V]HSSF
SPVRDNIQL[Label:13C(6)15N(1) (L)]
TEINR[Label:13C(6)15N(4) (R)]LPSA
TEMEKIRV[Label:13C(5)15N(1) V]C
SEVMGTTL[Label:13C(6)15N(1) (L)]
TPAEVSIVVL[Label:13C(6)15N(1) (L)]
RYGSFSVTL[Label:13C(6)15N(1) (L)]
SLAVVSTQL[Label:13C(6)15N(1) (L)]
QDFSVPQL[Label:13C(6)15N(1) (L)]
TY[Label:13C15N(1) (Y)]PKSGTTW
GLLQVTGVTRV[Label:13C(5)15N(1) V]
SPASDAYIV[Label:13C(5)15N(1) V]F

This will result in you getting all of the peptides that you have in "sample2.sky".

If you want to see what the Modified Sequence is for the precursors in your document, you can use the Document Grid.
That column is located at:
Proteins > Peptides > Precursors > Modified Sequence > Modified Sequence Full Names

Does this help, or were you hoping to be able to do something different?
-- Nick
 
lyjgbb responded:  2020-06-12
Hi Nick,

This is great! it works very well! I really appreciate your quick and professional recommendation here!
I can continue to rebuild the SIL library by the same way.
By the way, there are some typos in the list you generated. The V[Label:13C(5)15N(1) V] has to be V[Label:13C(5)15N(1) (V)] , otherwise it will get an error in Skyline.

Thanks!
John
 
lyjgbb responded:  2020-06-16
Hi Nick,

Another quick question:
Just like the way to insert SIL peptide, what is the amino acid format for inserting a peptide sequence with modified amino acids?
For example, a peptide TEMEKIRVC with cysteinylation on C?
Or the same peptide TEMEKIRVC with glutathionylation on C?


Thanks,
John
 
Nick Shulman responded:  2020-06-16
It would be the same syntax. The thing that you put inside of the square brackets is the Name of the modification. The modification can appear in either the "Structural Modifications" list or the "Isotope Modifications" list at "Settings > Peptide Settings > Modifications".

This is the webinar that we point people at when they have questions about managing the lists of modifications in their Skyline document. (I have not watched this webinar, but I have heard that it helps).
http://skyline.ms/webinar10.url

-- Nick
 
lyjgbb responded:  2020-06-16
Thanks a lot again! That webinar is helpful.
John