Normalization by TIC

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Normalization by TIC caminha marcelle  2021-10-22
 

Hi,
How the normalization of PRM data by TIC is calculated?
Thank you in advance.

 
 
Nick Shulman responded:  2021-10-22
When Skyline extracts chromatograms from a raw file, Skyline also looks at the TIC chromatogram that is present in the raw file. Skyline only looks at the points along that chromatogram corresponding to the MS1 spectra. Skyline integrates the entire length of that MS1 TIC chromatogram in order to find the TIC Area for that raw file.

There is a column in the Document Grid "Total Ion Current Area" which will tell you what the TIC area was for any particular replicate.

When Skyline needs to normalize a particular value by TIC, Skyline divides the value by the TIC Area for that replicate, and then multiplies by the median TIC Area across all of those replicates. For this reason, the area normalized by TIC will usually be a number which is very close to the non-normalized area.
-- Nick
 
roman sakson responded:  2021-10-24
Hi Nick,

I thought that this might fit in this thread. First of all, great feature, I like it a lot, especially in combination with visualization in Panorama! I would like to understand it a bit better:

1) In Skyline, I can choose not only "TIC" but also "Base Peak", however I am always getting the message "No base peak chromatogram found" for my MS1 Filtering Full Scan results. Am I doing something wrong?

2) Just out of curiosity, I noticed that when I manually integrate all MS1 spectra in the Xcalibur Qual Browser from Thermo, I am constantly getting 60-fold higher TIC areas than in Skyline. This is a constant factor for several raw-files that I checked, so might have something to do with how signal intensities are being processed (TIC chromatogram in Skyline has the same overall appearance compared to Qual Browser but the intensity values seem a bit lower). Do you know what is going on there?

Thank you,
Roman
 
Nick Shulman responded:  2021-10-25
Roman,

The only way to see the base peak chromatogram in Skyline would be to open a document where the chromatograms were extracted using a version of Skyline which is version 19.1 or older.
Older versions of Skyline would extract the base peak chromatogram and the TIC chromatogram. This involved looking at every MS1 spectrum.
Starting in 20.1, as an optimization, we changed it so that Skyline would use the TIC chromatogram that is typically present in raw files. At the same time that we made that change, we also made it so that the Base Peak chromatogram never gets stored in the .skyd file.

The Total Ion Current Area value that is reported in Skyline is 60 times smaller than what it technically should be. The Skyline code that integrates that particular chromatogram does not multiply by the conversion from minutes to seconds.

We should probably hide the Base Peak menu item. It's too late (for backwards compatibility reasons) to fix the total ion current area discrepancy.
-- Nick
 
roman sakson responded:  2021-10-25
Nick,

great, thank you for making this clear! Would TIC extraction also work for other vendors than Thermo (Bruker timsTOF etc.)?
 
Nick Shulman responded:  2021-10-25
If Skyline does not find a TIC chromatogram in the file, then Skyline falls back to the old way of extracting the TIC chromatogram the by summing the intensities on every MS1 spectrum in the file.
In all cases, when you are extracting chromatograms from full scan data that has MS1 spectra, you should end up with a value in the "Total Ion Current Area" column in the Document Grid.
-- Nick
 
roman sakson responded:  2021-10-25
Thank you!