Dear all, I'm grateful for all the responses and I did find many of
the tips to be very helpful. The servers/programs that people mostly
come up with are listed below. I used MULDI-TOF and Artem has
suggested more accurate measurement. And also many people suggested
that I should combine N-terminal sequencing with the mass-spec to have
unambiguous identification. I was also suggested to cut out the two
bands from the SDS-PAGE for further trypsin digestion and another
round of mass-spec to help identify the bands. I'm grateful for those
experts that are always "on-call", even during the weekend, at this
forum, not only to my questions, but to many others. Please see below
for more details of the individual responses – thank you all!
Sincerely yours, -yong
1) http://prospector.ucsf.edu/prospector/4.0.8/html/msfit.htm
2) http://expasy.org/tools/peptidecutter/
3) http://bioinformatics.genomicsolutions.com/PawsDL.html
4) http://www.expasy.ch/tools/findpept.html
Subject: (bigger) fragment identification of limited proteolysis w/ mass-spec
------------------------
From: Yong Tang <[log in to unmask]>
Dear all, just a super dummy question: I treated a protein with
trypsin, found the protein being degraded into two well-define
fragments, ran a sizing column to find them co-elute, sent the peak
fraction for mass-spec, got the two masses. Now here is the question -
is there any program readily available for me to roughly identify
these two (around 20K) fragments with the full-length sequence and
these two masses, and of cause, with the fact that I use trypsin to
cut it? I checked a lot of programs listed on Expasy to find them
mostly dealing with smaller peptide length. Any information would be
highly appreciated. Thanks and have a nice weekend, -yong
--------
From: Jan Abendroth <[log in to unmask]>
Yong,
try the protein prospector (http://prospector.ucsf.edu/) and click the
MS-Digest button.
Then chose a small number of missed digests.
--------
From: Mark Brooks <[log in to unmask]>
try Protein prospector (it is listed at Expasy). The best is MsFit:
http://prospector.ucsf.edu/prospector/4.0.8/html/msfit.htm
It takes a while to learn how to use it. The protein has to be in one
of the databases also, but its very good once you can find it!
--------
From: Peter Cherepanov <[log in to unmask]>
To be really sure, run the digested protein (2-20 ug, the more the
better) on an SDS-PAGE, transfer onto PVDF membrane and ask someone to
do N-terminal sequencing of both bands (aka Edman degradation). This
will identify the N-termini, given more or less exact masses that you
already know from MALDI you will be able to map both fragments without
doubt. You can check our paper - PubMed #15371438 if you want a simple
example.
you can also cut the bands from your gel and try complete in-gel trypsin
digest/mass spec - you will see which peptides are present in which
fragment. These method can fail or produce confusing results, because
not every tryptic peptide will be detected by mass spec (inefficient
ionisation etc) so you will often not see the terminal peptides; and
there will always be conflicting peptides, found in both (due to very
high sensitivity of mass spec for some peptides).
Anything else will be really a guess work.
--------
From: Kornelius Zeth <[log in to unmask]>
there is. Depending on the prcision of the mass.
--------
From: Juan Sanchez-Weatherby (UEA) <[log in to unmask]>
Have a look at this site: http://expasy.org/tools/peptidecutter/
It will give you lots more than one site but it may give you an indication
of around where to look for the site.
--------
From: Oganesyan, Vaheh <[log in to unmask]>
You can input your sequence to Expasy server, choose tripsin as an
enzyme and see where the software will predict the cleavage. Compare
to your experimental data. On the experimental side the best would be
to send the cleaved products to N-terminal sequencing. Usually it can
read at least first 4-5 aa.
--------
From: ezra peisach <[log in to unmask]>
Don't know of a program for you to use - but end terminal sequencing will
tell you where the fragments start.... (assuming you can separate them on
gel native/denaturing - or an ion exchange column)...
--------
From: Wendy Gordon <[log in to unmask]>
Try this one from expasy; I used it for my 36kDa protein...
http://us.expasy.org/tools/peptidecutter/
--------
From: Jason Hurlbert <[log in to unmask]>
You might want to look at the program PAWS. It will do exactly what you
want, but it only runs under Windows. I've not tried it under WINE in
Linux, but it may work in that situation as well.
The link to the program is:
http://bioinformatics.genomicsolutions.com/PawsDL.html
--------
From: Anthony Addlagatta <[log in to unmask]>
Note that any program would only predict the cutting site based on
the primary sequence. What you are seeing is structure dependent. I
doubt if there are programs that are smart enough to do that kind of
job.
--------
From: Artem Evdokimov <[log in to unmask]>
There are several MS-specific programs that I know of but they're not in the
public domain. I have a couple of crude PERL scripts for this and an Excel
application written by a former colleague. These are too crude to send out,
but I would be happy to run your sequences and masses through them for you.
How accurately were the masses determined (hopefully using ESI LC MS and not
MALDI-TOF, since the mass accuracy of the latter is not as good)? Do the two
masses add up to the total m.w. of the starting protein? If they don't add
up this likely means that either a) there are smaller fragments that you're
not detecting or b) your original m.w. is not what you expect. I assume you
also ran the original protein MS in the same way as the fragments? Is the
original protein homogenous? If it is not this makes life quite difficult.
Finally, since the two fragments associate tightly enough to co-elute on
sizing this probably means that you've cut into an exposed loop or a linker
region between tightly associated domains.
Best regards,
Artem
--------
From: [log in to unmask] <[log in to unmask]>
did you think about to do (or send your samples) for the edman degradation?
--------
From: Meier Chris (SLH) <[log in to unmask]>
I would suggest you use the FindPept tool:
http://www.expasy.ch/tools/findpept.html
Just enter your protein sequence and the Mass-spec results
of your fragments: PindPept will then calculate all the
possible fragments which correspond to this mass.
Simply choose the correct one
given the known cleavage behavior of your protease
(trypsin).
Not the most elegant solution, but it works:
I have used it successfully for all my limited proteolysis
experiments.
--------
From: [log in to unmask] <[log in to unmask]>
There is a program called PAWS that does exactly what you're talking
about, and
I think there is a freeware version (unfortunately for PC only, it appears):
http://bioinformatics.genomicsolutions.com/paws.html
--------
From: Juergen Bosch <[log in to unmask]>
You should be able to solve your problem with the following link:
http://us.expasy.org/tools/findpept.html
--------
From: Paul Kraft <[log in to unmask]>
Certainly you can seperate these fragments by SDS-PAGE, cut the bands
out, redo the proteolysis and throw them into the MS again...
---------- Forwarded message ----------
From: [log in to unmask]
There is a program called PAWS that does exactly what you're talking
about, and
I think there is a freeware version (unfortunately for PC only, it appears):
http://bioinformatics.genomicsolutions.com/paws.html
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