Should Biognosys iRT FASTA be included in the MaxQuant database in order to work?

Should Biognosys iRT FASTA be included in the MaxQuant database in order to work? michelep  2019-02-28

Hi All,

I am doing some DIA analysis and recently bough the iRT Biognosys peptides to spike in for RT alignment.

My Library contains around 90 DDA runs and only 15 of them have actually the iRT peptides in.
ALL the DIA runs (10) instead have the iRT spiked in!
When I follow the DIA Webinar 14 for adding the iRT prediction etc, Skyline doesn´t find the iRT in any of the DIA runs, even tough I can see they are there from the Excalibur XIC.
Then when I do the mPhophet FDR calculation/peak picking the iRT prediction is greyed out.. and can´t be used for peak picking

Is this because the MaxQuant search I did to create the library does not contain the Biognosys iRT FASTA? So those IDs are NOT in the MSMS file?
Thanks in advance to anyone that can help me


Brian Pratt responded:  2019-02-28

Hi Michele,

We can reach a quicker understanding of your situation if I can see your Skyline document. Use the File > Share > Complete menu item and upload the resulting file to If you need to keep it private we can arrange something else.


Brian Pratt

michelep responded:  2019-03-01

Hi Brian,

thank you for replying really FAST!
Of course I can share the Skyline analysis, but since you are giving me the possibility, I would like to do it in privat.

Please let me know how we can arrange that and also if you prefer the skyline document before or after I apply the mPhopet peak picking algorithm using the decoy as background.


Brendan MacLean responded:  2019-03-01

Yeah. You definitely want to include the iRT peptides in your MaxQuant search. I am not quite clear on how you created your iRT library/calculator without including the iRT peptides in your MaxQuant search. This is where looking at your document will probably help. Do you have an iRT library containing all of your targeted peptides and the Biognosys iRT peptides as the standards?

Finally, "Skyline doesn´t find the iRT in any of the DIA runs, even tough I can see they are there from the Excalibur XIC." makes it sound like you are targeting the iRT peptides in your DIA runs, but Skyline is not extracting the same chromatograms you see when you perform this operation with Xcalibur. That just doesn't seem likely, though.

You could also post some screenshots of what you are basing these conclusions on and we might be able to offer more help. Though the files themselves will likely tell the whole story.

I'll send email about the private drop point.


michelep responded:  2019-03-01

Dear Brendan,

Thanks for the reply. I will defenetely repeat the MaxQuant search with a FASTA containing the biognosys iRT FASTA. I was just trying to avoid that since my library is obtained by 99 DDA :D

About my phrase "Skyline doesn´t find the iRT in any of the DIA runs, even tough I can see they are there from the Excalibur XIC." I explain myself incorrectly.. sorry...
Skyline does find all of them (please see attached picture), is just that does not use them, in any of the DIA runs for alignment.

I created the library (8400 IDs) by doing: build library-->load the MS/MS file from MaxQUANT, then View Spectral Library--> check Associate proteins and Skyline added 8400IDs in the assay and 137K peptides
After doing this, the iRT peptides were not there (since not in the MaxQuant FASTA search!) So I manually imported the iRT FASTA by doing: File-->import-->FASTA and selected the iRT FASTA downloaded from Biognosys website.
Then I imported my DIA runs by using 10min from MS/MS IDs since I couldn´t use the RT calculator.

I hope I answer your questions and explained myself better.

Anyway now I will share the full analysis with your private link (thanks)

michelep responded:  2019-03-04

Dear Skyline Team,

I uploaded the full Skyline document to the private link your provided me.

Hope you can understand better the iRT calculation issue I am having now.
Best regards and thank you in advance

Nick Shulman responded:  2019-03-05

Your iRT database only contains information for your iRT standards.

That is, if you go to:
Settings > Peptide Settings > Prediction > Calculator Button > Edit Current
you will see numbers in the upper grid ("iRT standards"), but nothing in the lower grid ("Other iRT values")

Because of this, when you are extracting chromatograms, first extracts the chromatograms for your iRT standards, and then Skyline does a linear regression between the iRT score in the database (a number between -25 and 100) and the retention time at which the iRT standards were found in the chromatogram. Skyline finds the slope and intercept that convert between iRT score and retention time.

However, since your database does not contain numbers for any other peptide, that slope and intercept does not end up being useful for anything.

Yes, you should go back to MaxQuant, and do a peptide search on your 15 runs that contain your iRT standards using a FASTA file that contains both your peptides of interest and the iRT standards.
Then, when you are creating a spectral library from these new results, Skyline will notice that you have iRT standards, and Skyline will offer to add iRT information to the spectral library that is being created.

Also, if you have a spectral library that contains iRT standards and peptides of interest, you can add them to an existing iRT library by going to:
Settings > Peptide Settings > Prediction > Calculator Button > Edit Current
and press the "Add" button at the bottom and choose "Spectral Library".

-- Nick
michelep responded:  2019-03-07
Thank a lot Nick for your kind and good explanation!

I will do as you said and hopefully I will manage to use the iRT peptide as well in my Analysis

Thanks for all your support