Retention Time Calibration for DIA-data

Retention Time Calibration for DIA-data yb77618  2019-05-17

Hi Skyline Team:

I have DIA-MS data and pre-built assay library, so I want to use Skyline-Daily for data analysis.

The assay library was loaded successfully, 11 iRT-peptides were chosen as standard for retention time calibration.

However, I found out the criteria for DIA-MS data loading was so stringent, 80% peptides were needed with a correlation of 0.99 in regression.

Is that possible for us to change the requirements for calibration? Like to calibrate by hands or lower the R-square value?

(I want to remove some iRT-peptides for better correlation in the regression, an alert popped up, see attached image)

Thank you in advance for answering my questions.


Brendan MacLean responded:  2019-05-21

Hi Wei,
This alert is telling you something very useful and that is that you are already starting with only 9 of the 11 iRT standards in your document. If you truly expect only 9 of the peptides to be present and that is why you are only targeting 9, then you should edit your iRT calculator and delete the 2 that you expect to be missing. You will stop getting this error and Skyline will be more tolerant in its calibration in your document. When your calculator contains 11 standards but you only target 9, then Skyline loses the ability to drop any other peptides to achieve 80% at 0.99 correlation. If there is even 1 outlier, then Skyline will be under 80% of the standards, since you are starting with only 81.8%.

This is the meaning of the alert you are getting. You should either target all 11 of your standards or change your calculator to contain only the standards you expect to see.

Hope this helps. Sorry for the confusion.