Dear support team,

I am attempting to load post-analysis internally calibrated Bruker timsTOF .d data files into Skyline and am encountering an issue with the representation of calibrated data.

For the same acquisition, I have two differently processed datafiles:
ZTB11_Boltje-O-glycan-S1-2uL_GA2_1_1564_uncalibrated.d (no post-analysis calibrated)
ZTB11_Boltje-O-glycan-S1-2uL_GA2_1_1564_calibrated.d (post-analysis internal calibration applied in Bruker DataAnalysis)

In DataAnalysis, the calibrated file shows the expected improvement in mass accuracy, with peaks calibrated to approximately 2 ppm compared to ~23 ppm in the uncalibrated data (example shown for m/z 675.2466 in DataAnalysis_cali_vs_uncali.png). However, when loading both the calibrated .d file into Skyline, the calibrated file does not reflect the post-analysis mass calibration. The observed m/z values in Skyline appear identical to those from the uncalibrated data, suggesting that the post-analysis calibration is not being recognized. I have included the Skyline document and associated files Calibrated_vs_uncalibrated_file_2025-12-16_14-42-39.sky.zip for reference. This leads me to suspect that Skyline is accessing the original acquisition data rather than the post-processed calibrated data.

Is post-analysis internal calibration applied in Bruker DataAnalysis supported when importing timsTOF .d files into Skyline? What are the steps required to ensure the calibrated data are correctly loaded?

Thank you for your help.
Agnes

accidently posted this message orginally to PanoramaWeb, Brendan replied with the following message
2025-12-17 05:14:55
Hi Agnes,
Please post this to the Skyline support board:

https://skyline.ms/support.url

That reaches the Skyline team more directly and you will get your best response there. They will still be able to access your data on Panorama.

I understand your issue. Slides 22-27 of Skyline Tutorial Webinar 15 describe a case where I got a Bruker dataset without post-analysis calibration and eventually worked that out and managed to get the same dataset calibrated. That worked for me back then (2016), but it wasn't even timsTOF data. So, something may have changed more recently.

Unfortunately, the most obvious developer to look into this is on vacation until December 29. I hope one of us can still look into this, but it can certainly get attention within 2 weeks.

Thanks for taking the time to report your issue.

--Brendan