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, 

Similar questions and discussions