MSstatsTMT with Phospho-enrichment

[Talia Head]

I have a TMT-10plex labeled total proteome sample split into 8 fractions, and a subset of the same sample run through phospho-enrichment as just one sample (completed before fractionation of the total proteome, no subsequent fractions of the phospho-enriched sample).

In the annotation file, do I list the run from the phospho-enriched sample as fraction 9? It is from the same mixture as the global proteome.

Or should I list it as a second mixture, even though it is technically the same, in order to compare the phospho data to the total proteome?

I have two different conditions within the 10-plex samples and am mostly interested in comparing phosphorylation between the two conditions.

Thanks for any advice!

[Devon Kohler]

Hello,

This is actually a unique case that MSstats is not designed to handle; you will probably need to do some manual work to do the analysis.

You could certainly treat it as a 9th fraction, in this case the MSstats converter would just include all the phospho-enriched peptides with the total proteome samples. If you want to model the PTM, you would need to go into the data (post-conversion) and indicate which values are phospho PTMs and which are total proteome. From there you could separate the PTM/proteome into the format that MSstatsPTM takes (where we do total proteome adjustment) or just continue with the normal MSstatsTMT pipeline (without total proteome adjustment).

Hope this makes sense. 

Devon

[Talia Head]

Hi Devon,

Thank you so much for the input! I am going to try both methods to see what I can get to work and I will update here in case anyone in the future runs into this.

Cheers,

Talia

[Sam Siljee]

Hi Talia and Devon,

I'm also in a similar situation;

- One mixture, TMT 18-plex

- 8 fractions done for the proteome with 5 - 7 technical replicates

- Same mixture enriched for phosphopeptides, not fractionated, with 3 technical replicates

I would like to use total proteome correction, can I achieve this by changing the protein name to include the phosphorylation site after the `dataSummarizationPTM_TMT` call?

Sam