Hello, I am currently analyzing some phosphoproteomics data, but I have peptides with multiple phosphorylation sites or phosphorylations together with carbamidomethylation or oxidation. How can I handle this if I need each phosphorylated residue + site individually? How are the quantification values affected?
Thanks!!!
Hi Amanda,
you need to make some sensible decisions and stick to them throughout the analysis. We'll start with the easy ones; Cys-CAM should essentially be 100% for your Cys if is was done thoroughly. As such, it should not present any issues. By oxidation, I assume you mean methionine? Generally, that will be around the 5-10% level. If it's not, you've got sample handling issues, or something is happening in your test system. This could be real, or artefact, which you can hopefully determine.
In both cases, I recommend pooling the MSO (oxidised Met) version of the peptide with the unmodified version. Likewise for the Cys-CAM if you see the unmodified version.
For the phosphosites, again, you need to decide how to handle them. Unless you have evidence to the contrary, I'd be inclined to pool the versions of the peptide with a single phosphosite, regardless of what reside is phosphorylated. If you have good biology telling you different phosphosites lead to different outcomes, do not do this, keep them separate.
Likewise with multiple phosphosites in the one peptide. Pool unless you know they have different biological outcomes.
Even doing this, it very quickly becomes a complicated web. Be consistent make sure you tell reviewers what you have done when you get to that stage.
Hi Peter, thanks so much for your explanation. However, I still have doubts. I'm really new to this sorry. These are three examples of peptides and intensities from my data with characteristics I mentioned in my original question:
GPAPAPQQC(+57,02)S(+79,97)EPETK 6.915789127
EM(+15,99)LMEDVGS(+79,97)EEEQEEEDEAPFQEK 10.69081116
KRPQS(+79,97)PS(+79,97)PR 12.1003685
So from what you explained if I have many peptides from the same protein, I should choose the version of the peptide with a single phosphosite. But I often don't have that version. Instead, I have the peptide with multiple phosphosites (like example 3). In this case, should I choose one? what happens with the intensity?
For MSO and Cys-CAM, how should I proceed if I'm only interested in phosphorylation?
Thanks again for your help!
Hi Amanda,
if you've alkylated well, the vast majority of your Cys will be Cys-CAM, so treat that like plain boring Cys. ie. Cys-CAM is there and it's just another amino acid. You can check your level of alkylation by doing a search where Cys-CAM is a variable modification.
It's similar for MSO. That should be around the 5-10% of Met residues.
For this peptide:
EM(+15,99)LMEDVGS(+79,97)EEEQEEEDEAPFQEK
EMLMEDVGS(+79,97)EEEQEEEDEAPFQEK
Add the intensities to get one result.
Likewise if you see this version;
EMLM(+15,99)EDVGS(+79,97)EEEQEEEDEAPFQEK
Three versions of the peptide go into one result.
The hardest is this;
KRPQS(+79,97)PS(+79,97)PR
where you could also have
KRPQSPS(+79,97)PR
KRPQS(+79,97)PSPR
I'd treat the double phospho version as one result and combine the intensities for the two different monophospho versions.
I hope that helps.
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