Retention Time Issues for DIA data

support
Retention Time Issues for DIA data timofiiva  2019-06-24
 

Hello,

I encountered an issue with SkyLine recently and have not been able to sort it out:

  1. I imported the chromatograms from a phospho-enriched SWATH dataset and am analyzing the peak picking for my protein of interest
  2. One of the phosphopeptides of interest has a predicted retention time of 120 min
  3. For 5 of my 30 conditions, all is ok (screenshot1) where the peak is able to be picked by the expected retention time, and the chromatogram even extends further to 145min
  4. For the remaining 25, the chromatogram stops abruptly at 110 min (screenshot2). Even if you go through the Transition Settings/Full-Scan and then select "Include all matching scans", it will not allow you to see past the 110min.

I have tried the following so far:

  • To adjust the "explicit retention time" in the documents grid, and then reimport the file. What happens is that the "chromatogram information is unavailable.

Thank you in advance for the help!

Best,

Anastasia

 
 
Brendan MacLean responded:  2019-06-24

Can you possibly give us your Skyline document with data removed? (using File > Share - Complete to create a .sky.zip file) And one of the problem raw data files where you can't get a chromatogram past 110 minutes?

If you can upload these two files to:

https://skyline.ms/files.url

We can have a look and get a better understanding of what is going on. Even if we can just get a raw data file and the precursor m/z we can have a look at whether there are actually spectra that should match beyond 110 minutes, since the most obvious likely cause of what you are seeing is that somehow your mass spectrometer stopped collecting MS/MS spectra that match your precursor m/z at 115 minutes.

Have you tried having a look at your files using instrument vendor software (Xcalibur, Analyst, etc.) or the ProteoWizard tool SeeMS? Do you feel confident that there are matching spectra beyond 115 minutes?

--Brendan