Hej,
We have performed a TMT-10plex MS experiment (10 samples divided over 2 groups) to identify differently expressed proteins. We are discussing very much how to analyse the data.
What we do now (and what I believe is commonly done) is:
1. Relative quantification based on the label-reporter ions (sample 1 is set as 100% for scaling of each scan)
2. Mascot search for ID of proteins (using Proteome Discoverer)
3. QC: score >30, unique peptides >2, scan count>3
4. Median value of all unique peptides from a protein is taken as a "protein value"). All values of sample 1 are "1", as a consequence of the scaling.
5. Normalization to the median of all proteins in 1 sample
6. Statistics (T-test or Mann-Whitney U (due to low sample number, one cannot be sure about the normal distribution)).
According to a statistician, we are violating the "independent samples" assumption to perform a T-test. This is supposed to happen in step 1, where all absolute values are divided by sample 1 to get the absolute values of different scans within the same scale.
I cannot seem to find this in (proteome) literature. Could someone help us out? Are we violating the statistical assumptions or are we allowed to continue?
Thank you,