MS Amanda and TMT-10plex labeled data

[Carrie Chou]

Dear MS Amanda developer,

I am writing this letter for some help about the usage of MS Amanda.

I tried to use MS Amanda integrated in searchGUI 3.3.13 with my TMT 10-plex labeled data of 10 different mixed samples for identification of phosphorylation peptides and sites.

I gave an input file in "mgf" format, then set 'TMT 10-plex of K' and 'TMT 10-plex of peptide N-term' as well as 'Phosphorylation of S', 'Phosphorylation of T' and 'Phosphorylation of Y' as variable modifications, then I got the searching result in 'csv' format. but I don't know these phosphorylation sites come from which sample of 10 via the information provided by output file XXX.csv, because there is only information about monoisotopic mass of 229.162932, which is TMT-10plex modification mass, but no each reporter mass of TMT126, TMT127N, TMT127C, TMT128C, TMT128N, TMT129C, TMT129N, TMT130C, TMT130C and TMT131: 

So how can I know in which sample of 10 this phosphorylation site was identificated? Would you like to give me some information on how to do this? Is quantification of peptides and proteins necessary?

Looking forward to your reply. Thank you very much.

Best regards,
Carrie Chou

[Viktoria Dorfer]

Hi Carrie,

MS Amanda can unfortunately not tell you, which of your 10 samples have been phosphorylated, you need to do some post processing, i.e. quantification, on these results.

Here is why:

When you do TMT labeling the advantage is that a peptide that is present in multiple samples would end up in the same MS2 spectrum. So, the y1 ion for example in that spectrum is representing all y1 ions of that single peptide of your 10 samples (assuming all 10 samples have that peptide). The same holds for phosphorylated peptides, all samples that contain this phosphorylated peptide generated that spectrum. MS Amanda can only tell you which peptide is represented in a certain spectrum.

To know which samples contain the phosphorylated peptide, you have to look at the relative intensities of your reporter ions. A bit simplified this means, if all reporter ions are present your phosphorylation would be present in all samples.

As far as I know you could use PeptideShaker to post-process your MS Amanda results to learn which samples have a phosphorylation and which not.

Hope this helps!

Best regards,

Viktoria