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iRT issue chloe bardet6729  2018-01-18 07:14
 
Dear Skyline team,

I use the last Skyline version, but I also used the previous (3.7) and it doesn't work.
The MS I have is an Sciex5500QT

I would like to use an home made retention time calibrator containing 14 peptides and I have an issue to export correct calculated retention times.
When I use the 14 peptides to retention time calibration for 86 peptides from an LC gradient N°1 to an LC gradient N°2 (HPLC vs UPLC) using a basic linear regression, it works on Excel. However when I am using Skyline after reading the tutorial, I failed. Predicted retention times did not appeared in the "export transition list" and "calibrated retention time" that are noticed in the "export, report, peptide results" were not well calculated.

After the 14 peptides calculator built, I import results for the 86 peptides with gradient N°1 and I calculated iRT for the 86 peptides. This works, I had a great regression. Then when (in another skyline file) I import the results for the 14 peptides with gradient N°2 the RT predicted for the 14 peptides seems great, but I didn’t succed to export the RT predicted for the 86 peptide with the new gradient. When I wanted to export the transition list, Skyline asks me to choose the result file that I want to sort the retention times (or an average) never it talks about “predicted retention time” as you describe in your tutorial.

Thank you by advance for your help.
Best regards

Chloé Bardet
 
 
Nick Shulman responded:  2018-01-18 17:08
I am not sure that I understand your question. Can you send us your Skyline document?

In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including your iRT calculator and extracted chromatograms.

You can upload that .zip file here:
https://skyline.ms/files.url

It would probably be helpful if you uploaded both of the Skyline documents that you mention. Also, can you post of a screenshot of anything that looks confusing?
 
chloe bardet6729 responded:  2018-01-19 00:55
Thanks a lot for your answer.

I upload 3 files :
RTbeads pur corresponding to the file containing calibrator
MultiplexStandardcalibrate coresponding to the file containing peptide of interest + calibrator in gradient 1
Multiplex standard coresponding to the file containing peptide of interest + calibrator in gradient 1 and calibrator in gradient 2.

From the last file I expected to export retention time for peptide of interest in gradient 2 but I did not succeed. In fact, when I export transition list, Skyline ask me what RT I wanted to export : from result 1 or result 2 or an average ? (see screenshot)

And when I went in File/export/report/PeptideRTresult / preview : the predicted RT did not corresponding to the one I obtained when I made a linear regression on Excel to recalibrate retention time and in that case peptide were outside of my thin windows (0.8 min).
Also, when I dropdown to a calibrator peptide : it appears that the calibrated time is an average od both retention time (gradient 1 and gradient 2) (screenshot 2). I am sure I made a mistake but I do not undersatnd were it is.

Thanks a lot for your help.

Best regards

Chloé Bardet
 
Nick Shulman responded:  2018-01-19 12:19
If you want Skyline to use your iRT calculator instead of the retention times where Skyline found the chromatograms in your results, then I believe you should uncheck the box "Use measured retention times when present" on:
Settings > Peptide Settings > Prediction

If you have that box checked, and you have more than one replicate, then Skyline has to ask you which one of your replicates you want to use. If you have only one replicate, then Skyline does not need to ask that question. That is why you see a difference between "mutlipexetstandard" and "mutlipexetstandardCalibrate".

If you do not have that box checked, then Skyline will use your iRT predictor for all of your peptides.
 
chloe bardet6729 responded:  2018-01-23 05:57
Dear Nick,

First of all, I want to thank you for your help about the issue I sent you last week. It works!

Also, I woulld like to introduce myself. I am an analytical development project manager in a French CRO specialized in targeted protein quantitation in MS. Thus, we daily use Skyline and we develop the READYBEADS technology. This technology is based on an hydrosoluble bead coated with standard molecules such as peptides.

Then, we developed the RTBEADS product with 14 peptides that could be used as retention time calibrator for LC-MS monitoring.

I just wonder how it could be possible to add RTBEADS at your scroll-down menu in the iRT calibrator settings. What would you need exactly to create this calculator? If we sent you an excel table with peptide list associated to their iRT, could it be enough?

Thanks a lot for your support
 
Brendan MacLean responded:  2018-01-23 11:58
Hi Chloe,
What would be ideal would be a Skyline document with measurements of your peptides in a sample with any of the other iRT standards we currently list. It would be ideal if you could measure as many transitions as possible, ideally as many as 6. This allows us to insert the peptide targets into a Skyline document with the best N transitions (where N usually is between 3 and 6) based on the user deciding the number they want.

Measuring with a known standard allows us to calibrate to the iRT-C18 standard scale defined in the original paper.

Let us know if you have any further questions. Otherwise, we look forward to getting your Skyline document and including your standard mix in our list.

--Brendan
 
xavier homo responded:  2018-02-13 07:05
Dear Brendan,

My colleague, Chloé, contacted you some days ago regarding the possibility to add our retention time calibration kit (RT Beads) in skyline iRT. An experiment was necessary on our side to be able to get a skyline document with our kit combined with another kit available on the market. Then we processed the experiment with iRT kit from Biognosys and please, find attached to this e-mail the corresponding skyline results files.
Each peptide of our kit are represented with their respective 4 best transitions.

If some additionnal information are needed, please feel free to contact us.
Best regards
Xavier
 
Kaipo Tamura responded:  2018-03-05 11:03
Hi Xavier,
Thank you for the files. I have added this to Skyline and it will be available in the next version of Skyline-daily.

Thanks,
Kaipo
 
chloe bardet6729 responded:  2018-03-19 02:02
Hi Kaipo,

Thanks a lot for your help.
Anaquant team looks forward to seeing the RTBEADS index on the Skyline next version.

Best regards,
Chloé