Quantifying labelling efficiency

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Quantifying labelling efficiency sudip ghosh20975  2020-06-24
 

Hi I have a set of reference peptides which I labelled with TMT0. I ran both the labelled and unlabelled samples in Fusion and wanted to evaluate the labelling efficiency in Skyline. How do I proceed?

 
 
Nick Shulman responded:  2020-06-25
I am not sure I understand your question.

One thing to keep in mind with Skyline is that Skyline only knows how to quantify things using chromatogram peak areas. Many people who use TMT want to quantify things using a single spectrum. (And, I believe, often that spectrum is an MS3 spectrum). Skyline does not make it easy to get those sort of numbers out of a mass spec data file.

I know some people do use TMT with Skyline, but I am not sure what sort of experiments they are doing.

If you would like to learn more about what Skyline is capable of, you should take a look at the tutorials.
The absolute quantification tutorial talks about looking at heavy to light ratios:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_absolute_quant

-- Nick
 
Brendan MacLean responded:  2020-06-25
People have used PRM to get reporter ion chromatograms in Skyline, but it is a very niche approach. By far more common is the single-spectrum approach which Nick described and which Skyline does not support.