On Calculating I-Traq Ratio

Question

[Woa]

Here is an worked out example of calculation of Normalized intensity ratio for i-TRAQ reporter ions by a commercial software Scaffold Q+

The reporter ion intensities from all the identified peptide spectra are used to calculate non-normalized reporter ion ratios. These ratios are then used to compute the media adjusted normalized intensities. For example, all the reporter ion intensities are multiplied by their respective median non-normalized ratio. Once these median adjusted normalized intensities are calculated, new ratios are computed

Though the rest of the steps are obvious, I'm not sure how the "Median adjusted Normalized Intensity" values are calculated at the second step utilizing Non-normalized ratios. Can anybody please elaborate?

Answer 1

[Craig]

I am not 100% sure because I don't use Scaffold, but I have a pretty good guess based on my experience with isobaric tagging data analysis software. Usually for iTRAQ experiments you make the assumption that across all peptides, the median ratio will be 1:1. In other words, a typical peptide will have the same abundance in both samples/conditions. So let's say you run an experiment and you actually see a median ratio of 2:1 for the 114:115 tags. It probably means that you labeled twice as much protein with the 114 tag relative to the 115 tag. So the normalization process means you go through and divide every 114 tag intensity by 2 to compensate. This would give you the "median-adjusted normalized intensities", which you could use to calculate the median-adjusted normalized ratios.

[Woa]

Thanks Craig for the explanation