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TIC from Skyline does not match observed TIC

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TIC from Skyline does not match observed TIC Markus  2023-10-06 03:20
 

Hi Skyline Team!

I am analyzing Orbitrap Exploris 480 PRM data with an MS1 scan in each cycle. When I export the TIC from Skyline ("Total Ion Current Area") the numbers do not seem to represent the real TIC. I also tried Export -> Chromatograms but it is the same problem (although the numbers are different).

Here is an example why it does not make sense. I have 4 samples and a blank in between but the blank has the same (or even higher) TIC.

Replicate Name Total Ion Current Area
sample1 6465287168
sample2 6461600256
blank 6506602496
sample3 6478496768
sample4 6461451264

When I check the raw files in Freestyle the blank is a flat line at 2E7 while the samples are 2E10.

What am I doing wrong? I would be happy to send files if needed.

Many thanks,
Markus

OS is Windows 11, running Skyline-daily (64-bit) 23.1.1.268 (c58f1f56e).
 
 
Nick Shulman responded:  2023-10-06 09:16
Can you send us your Skyline document and your raw files?
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

Files which are less than 50MB can be attached to this support request. Your files will probably be larger than that so you should probably zip everything up together and upload it here:
https://skyline.ms/files.url

Skyline usually calculates the Total Ion Current Area by looking at the TIC chromatogram which is in the .raw file.
The TIC chromatogram in the .raw file usually includes points from both the MS1 and MS2 spectra, so Skyline removes the MS2 points, and then integrates the remaining points across the retention times.
After I see your raw files I will probably be able to tell you exactly how Skyline is coming up with the numbers that it has.
-- Nick
 
Markus responded:  2023-10-09 01:08
Hi Nick,
Thanks for your support!
Since these data are from a customer I would prefer not to upload them via the URL but rather send you a download link. Please contact me via email or specify an email address where I could send the link.
Many thanks,
Markus