Question about iRT prediction in skyline

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Question about iRT prediction in skyline 16622080827  2020-11-15
 

I have used iRT kit(Biognosis)in my experiment and I have also detected all the iRT peptide in my data both in PD software or check mannually.But when I try to import the PD result, it always shows that RT prediction failed.Maybe some peptides are in low intensity.Faced with this problem,what can I do for this condition?

 
 
Nick Shulman responded:  2020-11-16
The peptide sequences of the iRT standard peptides in your peptide search results are different from what Skyline expects the Biognosis standard peptides to be.

These are the sequences that Skyline expects for the 11 peptide Biognosis mix:
LGGNEQVTR
GAGSSEPVTGLDAK
VEATFGVDESNAK
YILAGVENSK
TPVISGGPYEYR
TPVITGAPYEYR
DGLDAASYYAPVR
ADVTPADFSEWSK
GTFIIDPGGVIR
GTFIIDPAAVIR
LFLQFGAQGSPFLK

Your PowerPoint includes a screenshot from some other tool, but those sequences have the amino acids in a slightly different order
LGGNETQVR
AGGSSEPVTGLADK
VEATFGVDESANK
YILAGVESNK
TPVISGGPYYER
TPVITGAPYYER
GDLDAASYYAPVR
DAVTPADFSEWSK
TGFIIDPGGVIR
GTFIIDPAAIVR
FLLQFGAQGSPLFK

For this reason, Skyline was not able to find any of the iRT standard peptides in your peptide search results.

Do you have any idea why your peptide sequences are different from what Skyline is expecting?

-- Nick
 
Brendan MacLean responded:  2020-11-18
I will just note that the official sequences provided by Biognosys:

https://biognosys.com/media.ashx/irt-kit-reference-sheet.xls

And which we use slightly modifies the true peptide sequences, presumably to make it more difficult for others to reproduce. Though, a mass spectrometrist in our lab once took this as a challenge and worked out the true peptide sequences. Perhaps this is the case here as well. But, if you use the true sequences in your Skyline document, then you also need to use them in your iRT calculator so that Skyline knows how to match them.