Mascot 13C button

[Jon H Man Uni]

Just wanted to get some feedback on how Scoffold handles data
generated using the Mascot 13C button?
As far as I understand this button allows you to keep database search
tolerances low whilst still allowing the identification of peptides
selected with non-monoisotopic peaks.

I was wondering how Scaffold dealt with such search results, so i ran
some tests and Scaffold seems to cope ok. Basically you get better
quality data for no extra cost i.e. the peptide/protein probability
scores are higher for the same dataset.

Is this the experience of other users?
and can someone from the company (or Matrix Science?) verify that this
is indeed the case?

Regards,
Jon

[Jason]

Hello Jon,

The protein and peptide probabilities of Scaffold depend on the Mascot
scores. If you do the search using Mascot with the 13C button selected
then Mascot's scores reflect the 13C isotope, and Scaffold's
probabilities for 13C IDs are increased based on the higher scores.
The only extra cost that I can think of is increased search time. If
anyone else has questions about this I would like to hear them.

Best regards,

Jason

On Mar 16, 9:56 am, Jon H Man Uni <j.d.humphr...@manchester.ac.uk>
wrote:

[Jon H Man Uni]

Jason,
Thanks for the prompt reply.
From what you say it seems to me that Scaffold treats the 13C isotope
as a variable modification but this doesn't appear as such when you
set up the Scaffold search.
If it is a variable mod then usually this results in an elevated cut-
off value for significance in Mascot and hence a general lowering of
peptide probabilities. Again this doesn't fit with my experience so
far as we see the opposite effect with our peptide probabilites and
Mascot cut-off values using the 13C button.
Cheers,
Jon

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[Jason]

Hello Jon,

Scaffold labels the 13C peaks as if they have a modification, but
other than that there is no penalty for 13C peptides. To my knowledge
Mascot does not penalize such identifications. Because Scaffold's
probabilities are based on Mascot's scores, and Mascot does not
penalize 13C IDs, there is no penalty for 13C IDs in Scaffold. Please
let me know if I misunderstand your question.

Best regards,

Jason


On Mar 17, 4:40 am, Jon H Man Uni <j.d.humphr...@manchester.ac.uk>

[Jon H Man Uni]

Jason,
Yes I guess I wasn't very clear with my last post.
I think you have mainly answered my query though - scaffold imports
peptide scores (and hence probabilities) from Mascot.
I cannot, however, actually see any 13C peptides ever highlighted as
modifications in Scaffold. The quickest way that I can find them is if
I look in the peptide lists and sort by peptide tolerances. This is
obviously different to other modifcations that are highlighted by
colour or selected from the drop down list in the samples window.
How Mascot actually deals with this 13C option is still a bit of
mystery to me though. Anybody fancy trying to explain?
Cheers,
Jon

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