Dear Brendan,
I have a question regarding DAT files created with Mascot Error Tolerant search.
When working with the MS1 full scan filtering wizard , I usually get a dialog box with all the modifications found in my DAT files. In my current project, I am working with DAT files created with Error Tolerant search in Mascot.
The DAT files seem to be correct and contain all the desired modifications in Unimod format.
Unfortunately when I import these data, I don't get any message to add 'guessed' modifications.
Furthermore, when I inspect the library in the explorer, I get a message that asks me to add only Carboxymethyl[M]=58
Can you tell me if I am missing some steps?
Am I wrong if I think that Skyline should automatically recognize (and ask me to import) the Unimod modifications in my DAT files (as in the MS1 tutorial)?
Might there be a specific problem with error tolerant searches?
Thanks for your support,
Salvo
If you want to inspect an ErrorTolerant DAT files, I could send you one by WeTransfer or similar ... |
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| Kaipo Tamura responded: |
2014-06-10 13:18 |
Hi Salvo,
Is it possible that you already have the expected modifications added in your peptide settings?
If that is not the case, please email me the file and the modifications you are expecting to see at kaipot@uw.edu (you can send it using WeTransfer or I can send you FTP server information).
Thanks,
Kaipo |
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| Brendan MacLean responded: |
2014-06-10 13:24 |
Hi Salvo,
Well, one possibility is that you already have the necessary modifications defined, which would allow Skyline to guess the modifications on your peptide sequences without this message. Are you also seeing that Skyline does not actually guess peptides for your matches? This is indicated by the peptides sequences lacking a peptide icon to the left of the text.
It would certainly be surprising, if Skyline did not show some sort of message for any modification it cannot understand. It should either state a possible Unimod modification or just that it cannot understand, say M[58]. Otherwise, it should guess your modifications, unless for some reason, your settings make them impossible to achieve.
In order to provide you the best support, I would need to see screenshots of the Library Explorer showing what sequences are failing to match to modified peptides, and even the tip text (hover the mouse of the sequence) of an unmatched and modified sequence.
Unfortunately, the information you have supplied is not enough for me to say much more. Hope this was somewhat helpful. If not, I look forward to hearing and seeing more.
--Brendan |
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| scappadona responded: |
2014-06-10 14:16 |
Dear Kaipo and Brendan,
Thanks for your prompt reply.
I have actually defined only the 'standard' modifications: - Carbamidmethyl(C) as fixed and Oxidation(M) and Acetyl (Prot N-term) as variable.
I think these are the only ones recognized by the import wizard, while I miss all the <pep_var_mod>.
I attach a screenshot of a peptide that I see both normal and modified in Mascot, and that appears only as unmodified in the Library Explorer.
I will also send you a Dat file, with corresponding raw file via WeTransfer.
Thanks again for your support,
Salvo |
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| Kaipo Tamura responded: |
2014-06-11 09:45 |
Hi Salvo,
When using your files, Skyline recognizes the modifications: "Acetyl (N-term)", "Acetyl (S)", "Acetyl (T)", Carboxymethyl (N-term)", and "Oxidation (M)". Are there more modifications that you would expect to see (if so, which ones)?
Thanks,
Kaipo |
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| scappadona responded: |
2014-06-11 10:54 |
Dear Kaipo,
Since we performed an Error Tolerant search, we actually expect hundreds of modifications.
If you inspect the DAT file, they are reported as as umod modifications, right at the beginning of the file.
Here you get a list of peptides and their variable modifications found (with Progenesis+Mascot Err Tol) in the same raw file.
Thanks a lot,
Salvo |
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Screenshot.pdf
Expected modifications.csv