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TIC not importing/displaying correctly thicks  2019-06-26 07:14
 

Hi all,

I am trying to do a simple import of MSe Qtof data with lockmass correction. I've never had an issue before, but now I have several .raw file that import the last third of the chromatogram incorrectly. Up to 80ish minutes looks as expected but something bizarre happens after that. I've attached what the TIC looks like from the acquisition software. Is the file corrupted? Any ideas?

Thanks,

Terry
 
 
Brendan MacLean responded:  2019-06-26 11:10

Hi Terry,
Could you possibly install ProteoWizard and then open the raw data with the SeeMS program that it installs? It would be interesting to know:

  1. Whether the TIC it shows looks like the one from MassLynx, or the one in Skyline, or something entirely new.
  2. Whether you see any evidence of low energy spectra near the times where the TIC is so incredibly high that would explain that much signal.

Also in Skyline, it might be interesting to look at the BPC for this file to see if it has a similar issue, indicating some very large peak in a spectrum or not, indicating a lot noise signal in a spectrum caused the TIC to be so high.

Let us know what you find. Thanks for the informative screenshots.

--Brendan
 
thicks responded:  2019-06-26 11:49

Thanks for the response! The SeeMS TIC looks the same as MassLynx TIC. The skyline BPC matches the Skyline TIC observations. For the spectra, after scan 4663 (83.9 min) none of the MS1 spectra show in SeeMS. (MSn do show). See attachments.

Thanks again,

Terry
 
Brendan MacLean responded:  2019-06-26 11:59

Hi Terry,
Can you ZIP the file and upload it for one of the Skyline developers to look at?

https://skyline.ms/files.url

The SeeMS TIC (and probably the MassLynx TIC as well) are pre-calculated and stored in the data file, while the Skyline TIC and BPC get calculated from scratch during chromatogram extraction from the spectra stored in the raw data file.

It seems like there must be something odd about the MS1 spectra in the file that does not match the stored TIC.

Thanks for the new screenshots.

--Brendan
 
Matt Chambers responded:  2019-06-26 12:27

Do you get an error in SeeMS when you click on those scans past 4663? Otherwise I'm not sure what you mean by "show" since your screenshot lists a full table of MS1s after 4663.
 
thicks responded:  2019-06-26 12:46

I should have been more specific. The MS1 do not display in SeeMS, though the scan data is listed. No error is shown when I try to display; it's just blank. Uploading the .zip now. Thanks for all the input!

Filename: 190624_SMART_NIST-UV_TCEP-0-5_1.raw.zip
 
Matt Chambers responded:  2019-06-27 13:24

Can you view the spectra from MassLynx? I'm not sure if this is file corruption or what, but like you said it's not giving an error, just empty spectra (even though they report having non-zero TIC).