Re: Protein Sequencing and MS
Laurey Steinke (lsteinke@molbio.unmc.edu)
Fri, 27 Feb 1998 10:19:15 -0600 (CST)
Dear Jim,
There aren't very many labs out there doing sequencing of unknown proteins
by MS alone. There are a few, Roland among them, who are doing great
things. MS data is not easily read--I think in fact that one would need to
go the direction of Ken William's lab at Yale and hire someone with a PhD
in MS (preferably trained by Roland or someone like him) and then you could
limp along behind, and maybe figure out how its done. I don't have a MS
that can do any sort of sequencing, so I do not get to see the data on a
regular basis.
I have friends that are sure that MS is going to change (has already
changed) everything. They also run Edman sequencers. I gave them a
hypothetical option: 1 picomole or less of a 25mer peptide that you needed
the sequence of unambiguously. You are fairly sure it is not in the
databases. It took someone 1 year to produce. Do you put it on your Edman
or on your MS? (And putting it on both wasn't an option, real world 1/10
on the MS and the rest on your Edman is probably the best option--although
once again, protein sequence from a 100 femptomoles is something few seem
to do well.) Anyway, my friends would all put the peptide in for Edaman
degradation.
Edman can tell the difference between isoleucine and leucine, MS/MS and
PSD can tell you more about where the phosphates and the glycosylation
sites are, and give you some sequence data, even, apparantly, in the hands
of someone without a degree in interpreting MS data. If you can afford
both, get both and use them in tandem. I can't just now, so I nurse the
protein sequencers and baby them along. (They are both working well
today, knock on wood.) Someone else will need to speak for the reliability
of MS instruments...my Maldi-tof is a real lemon, and works correctly much
less often than the sequencers.
Now, all of you who sequence unknowns on your MS instruments only can
write in and be counted.
Let the mad rumpus start!
-laurey
Weather inconsequential: Snowing just now. Our flirtation with spring has
ended. Lets hope the daffodils and tulips (up early this year) survive.
Hope all of you on the coasts are hanging in there!
Laurey Steinke
Protein Structure Core Facility
University of Nebraska Medical Center
Omaha Nebraska, 68198-4525
Phone (402) 559-6647
FAX (402) 559-6650
lsteinke@molbio.unmc.edu