Normalization DIA TIC

Normalization DIA TIC dkueltz  2015-03-16
Hi Brendan,
Thanks for the 3.1 release! I was wondering whether there is a way to normalize label-free data against the TICs of DIA MSMS runs (I get those using Bruker DataAnalysis). I have entered a separate column in the Skyline replicates table with the relative TIC integrals for each replicate. But I cannot select that column for normalization under group comparisons. It only shows up as an option for control group annotation and identity. It would be nice to have the option to select this column of DIA TIC values for normalization. I have been using the DIA TICs to normalize each transition in a sample/ replicate-specific fashion to account for small differences in loading or sample ionization after exporting the data to Excel. But with the new group comparison that includes statistics it would be great to be able to do it within skyline to save time.
Thanks for any suggestions,
Brendan MacLean responded:  2015-03-16
Hi Dietmar,
Interesting idea. Skyline does now store the TIC chromatogram for full-scan experiments. I am not that familiar with this mode of normalization. Are you saying you just sum up all intensity measures in the TIC, which seems like it would be dependent on sampling frequency, or are you taking an area under the curve of the entire gradient? Or something else?

I would like to understand better. It seems like we might be able to help, either by offering this as a choice, or, as you suggest, by allowing you to normalize by a replicate annotation value, which also seems reasonable.

Thanks for your feedback.

tobias.kockmann responded:  2019-09-04
Hi Brendan, Hi Dietmar,

I also have a case here where a TIC-based standardisation might be beneficial. The data was acquired using PRM incl. heavy std. peptides. By looking at the TIC I actually realised that the total sample amount was not properly adjusted before digestion (TIC area shows a high fluctuation from sample to sample, std. peptide signals are very stable).

Does anyone have a reference how TIC-based standardisation should be performed in this case? I was thinking to export to MSstats and do this manually prior to dataProcess(...) by scaling each light signal to def. unit of TIC area. As Brendan pointed out the TIC total area is reported by skyline. Does this have any downstream affects on statistical testing?

Nick Shulman responded:  2019-09-05


If you want to do TIC normalization in Skyline, you can take advantage of the feature where Skyline lets you specify the "Explicit Global Standard Area" to use for each replicate.

Here's the instructions from

When Skyline extracts chromatograms from MS1 data, Skyline remembers the total ion current.
You can see this value in the Document Grid.
It's under:
Replicates > Files > Total Ion Current Area

This column will contain the integral of the Total Ion Current of all of the MS1 scans.

Right next to the Total Ion Current Area column is the "Explicit Global Standard Area" column.
If you want to normalize to an arbitrary number, then you can type that number into the Explicit Global Standard Area column. Then, whenever you ask Skyline to normalize to Global Standards, Skyline will use that number instead.

A common thing to do is to copy the values from the Total Ion Current Area column and paste them into the Explicit Global Standard Area column.

(It has been suggested that we should add "TIC" as one of the normalization methods along with "Global Standards", "Equalize Medians" etc.)