At 17:02 09.05.00 -0500, you wrote:
>As my question will make clear, I am a novice user of mass spec.
>
>Presently I am working with yeast proeins. We cut a band of of a gel
>and digest it with trypsin, make measurements of fragment masses via
>MALDI and then use protein prospector to idetify the ORF. I understand
>how all of this works (more or less).
>
>But what happens in organisms with lots of introns? Does the program
>predict the splicing sites well enough to get a reasonable approximation
>to an ORF. That is, can I hope to do the same things with organisms
>with lots of introns that I co with yeast?
>
>Thank you for your help.
>
>Danny Kohl
Dear Daniel,
what you are worrying about is a general problem in identification of
proteins by MALDI peptide finger print only. What you are doing is to
identify a protein by matching masses of peptides versus the sequence in
the database. This works well for protein databases or DNA databases which
contain no introns. The problem arises if your databases contains only
EST's or all the introns as well or if the organisms DNA is not completely
sequenced. In these cases a PMF is of only limited use, you will need
"real" sequence information either from MALDI-PSD or ESI-MS/MS experiments
because you simply won't find a good fit of a peptide finger print against
a 50 amino acid stretch (maybe you'll get good sequence coverage but many
unassigned masses).
On the other hand, with sequence information you can possibly close
sequence gaps by assigning the intron and exon sequences (have a look at
http://www.pil.sdu.dk/ASMS_Contributions.htm from M. Mann et al, especially
at "Identifying proteins in genome databases" and "Advances EST
searching"). Also you can identify proteins by homology to other organisms,
which will work with PMF only in cases of very high similarity.
Yours sincerely,
Marcus Macht
**************************************************************
Dr. Marcus Macht
University of Cologne
Centre for molecular medicine - Service laboratory
Joseph-Stelzmann-Str. 52
50931 Cologne, Germany
Tel.: +49 221 478-6995
Fax: +49 221 478-6977
e-mail: Marcus.Macht@uni-koeln.de
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