one final comment(s) from my side before I shut my mouth. My comment that
MSMS data are bad to decipher the sequence of an unknown peptide can
certainly be challenged. But why then has the identification business
become so popular? Because MSMS data are so hard to interpret, whereas
matching something to a known is far simpler. This is why these programs
are so successful. I totally agree with Ioannis' comment that it requires
an enormous amount of ability to go through REAL interpretation of MSMS
data, and I certainly didn't mean to be unfair to those who are real
experts in this. When we got our instrument we had to go through this and
it took us an enormous effort to acquire the appropriate knowledge. But
because we went the hard route, I know what I am speaking of.
Let's face the facts: the quality of a CAD spectrum depends on the
collision energy. I an automated run where an instrument is switching from
precursor scanning to daughter scanning there is hardly enough time to
optimize the collision energy. It is usually fixed to a certain value
which is supposed to be optimal for a given peptide length. When we run
digests through our instrument we get good spectra (I mean spectra which I
would say they can be easily interpreted manually) from about 3 to 7
peptides. This is more than enough for identification purposes. But if you
would want to obtain sequence information from all the others, you'd have
to optimize the collision conditions individually for each peptide. The
other thing is that one usually reads a short stretch of 3 to 4 amino
acids in a CAD spectrum. Fine for identification purposes! But tell
somebody to devise an oligo on such sequence data: you'd be killed!
Going in depth through sequencing really requires the approach Rich
Johnson was mentioning: the methylation procedure pioneered by Don Hunt.
But given the time scale research is being conducted these days and given
the limitations in terms of amount scarce proteins are available these
days I do not think that this is a viable approach.
Again, I appologize for too harsh comments, I do know how much experience
there is in the world of CAD spectra.
Regards
Paul