Another "Chromatogram information unavailable' problem

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Another "Chromatogram information unavailable' problem SanL  2019-05-24
 

Hello,

I already saw lots of posts about this issue but I felt like none of them really solved my problem. I apologize if I missed the one that did.

I am new to Skyline like most posters. Some information:

Object: I want to build a spectral library for OpenSWATH use
DDA files: 29 .group files (ProteinPilot), each with Biognosys-11 iRT peptides
Background proteome: Whole SwissProt+iRT fasta

Afterwards, as "results" I loaded in the wiff files of these 29 DDA runs in again. The import went well, I can see TIC and BPC of each. However, for transitions I only get "chromatogram information unavailable" message.

I feel like this issue has a really easy solution and I am just missing something very basic. I sadly cannot figure it out at the moment though. Do you have any idea?

I will add some settings I used below and uploaded the Skyline.zip file as well as a zipped .wiff file in case those are of any help. [EDIT: Files apparently too big to upload]

Thank you so much in advance!

Best,
Sanni.

(Some additional relevant information if necessary:

Peptide settings

  • Prediction: Is set, time window at 10 mins

Filter transitions:

  • Precursor charge: 2,3,4
  • Ion charge: 2,3
  • Ion types: y,b
  • Product ion selection from ion 3 to last ion-1
  • ion match tolerance 0.5
  • Pick 6 product ions
  • 3 minimum product ions
  • from filtered product ions
  • Full-scan set to TOF, 30.000 resolution, use only scans within 5 mins of MS/MS IDs )
 
 
Brendan MacLean responded:  2019-05-24

So, you imported your DDA data into Skyline after building the library? Usually, we would recommend importing DDA data with MS1 filtering only and using ion type "p" instead of "y, b". It sounds like you are instead treating your DDA data like it was DIA or PRM data. What type of MS/MS filtering are you using Targeted, DIA or DDA (available only in Skyline-daily)?

Almost always you would want "p" in your Ion types field when dealing with DDA data. People do sometimes target the fragment ions in DDA data. Though they are not quantitative and will probably just mess things up unless you use Skyline-daily and the DDA setting for MS/MS filtering. I never do this myself when looking at DDA data imported for library building. I always use just "p" and MS1 filtering.

Hope this helps. Thanks for including so much of your settings. Screenshots are often helpful as well and can be pasted into PowerPoint or Word and posted with your report.

--Brendan

 
SanL responded:  2019-05-24

Hey,

alright, I suppose I understood something very wrong then and also worded my first post badly too.

I want to use DDA runs (n=29) to build a spectral library for SWATH use in Skyline.

I did that by selecting the settings mentioned above in "peptide settings" and "transition settings". Subsequently, in "peptide settings" -> "library" -> "build library" -> "iRT standard peptides" set to the biognosys 11 as mentioned above. Input files are 29 .group files. Afterwards, I uploaded the SwissProt.fasta, filtered a bit more with "refine".

I just downloaded the Skyline-daily and redid the whole thing with MS/MS filtering for DDA. I didn't know Skyline offered that, thank you for pointing it out.

"So, you imported your DDA data into Skyline after building the library? Usually, we would recommend importing DDA data with MS1 filtering only and using ion type "p" instead of "y, b". It sounds like you are instead treating your DDA data like it was DIA or PRM data.

As you could probably tell, it works well if I use precursor filtering only. I figured I could look into DDA data with that as well but... really, in retrospect I didn't question what I was trying to do too much, I apologize for having bothered you due to my mindlessness there.

Most importantly though; thank you so much ! I'll play around some more.