Urgent question: protein sequencing.
peteashton at NOSPAMhotmail.com
Thu Sep 12 03:34:24 EST 2002
The best approach depends on the organism that the protein comes from, or
rather, how much sequence information there is. The quickest approach would
be to digest the protein with trypsin and get a peptide mass fingerprint.
If the full sequence of the protein is in GenBank (or one of the other
databases), then this will usually give a rapid and firm identification.
If that fails because the sequence is not there, then using the same set of
peptides from the digest, it should be possible to do some kind of MS/MS. I
don't know how much you know about this stuff, so forgive me if I am
patronising you. MS/MS lets you select an individual peptide (without all
the hassles of HPLC) and look what fragments are generated after collision.
Generally, most facilites now use an LC on the front end of the instrument
to improve the number of peptides that can be detected, and they usually
rely on an electrospray machine (such as a Q-ToF or QStar). However, it is
also possible now to do the same thing on a MALDI-ToF-ToF instrument. There
are two things you can do with the data. You can try and interpret the
spectrum to give a "de novo" peptide sequence. Getting a couple of these
that come from the same protein will usually be enought to convince a
reviewer that you have identified the correct protein, but this can take a
lot of effort.
Alternatively, you can just submit a list of the fragment masses to a search
and it will tell you if there are any peptides in the database that would
same pattern of fragmentation. The beauty of this is that it will match to
EST databases as well as to full length proteins, and that is probably
enough to give you and idea of what your protein is.
"SK" <pianoman at mindless.com> wrote in message
news:7o7qnukmt8u1h48qqs0tr99kv5v87rg2td at 4ax.com...
> Hi there!
> I have a question. I pulled down unknown protein by affinity
> chromatography. I would like to identify what it is.
> What method should I use?
> Mass spec or N-terminal sequencing?
> What should I do if this protein is blocked at N-terminus? Is there
> any method to overcome this problem to do N-terminal sequencing?
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