Re: peptide sequencing

Deb McMillen (mcmillen@morel.uoregon.edu)
Tue, 15 Jun 1999 09:42:18 -0700 (PDT)

Jeni, Is your "new" instrument another old 477--or is it a 494? This
sounds a lot like a problem Sandie from Texas reported last week --I
wonder if they are related? If they are, it might actually point to a
chemical problem and maybe you could both compare notes on lot numbers of
reagents used. Otherwise, it is just a coincidence that you have the same
instrument problem--but it could be due to the same thing. One thing I
wonder about is is the sequencer fine when it starts out and then as you
progress through several cycles you have a venting problem that prevents
delivery of something somewhere in the system, hence lousy ry. Is the
size of your injections the same as you proceed? Does your ry pickup
again each time you restart a sample? So some line is vented that isn't
vented from run to run? If you run many standard cycles in a row do you
get the same phenomenon? If you stop this sample when ry is low and open
the system up, and restart does ry go back up? Have you watched
deliveries to be assured that TFA is delivered for the cleavage step?
Does the transfer to the flask look good? We've run, many years ago, all
150 oof the peptides that we made in our 470 (usually with one grain of
resin if possible) to check synthesis before cleavage--always seemed to
work for us then.

Hope this helps some,
I'll follow with a forwarding of Sandie Smith's email from last week,
Deb McMillen
Institute of Molecular Biology
University of Oregon
EUgene OR 97403

On Mon, 14 Jun 1999, Jeni Lauer-Fields wrote:

> Hello,
>
> We need a little help from the sequecing gurus out there. I am a relative novice
> at this so be gentle with me. Our lab has been running a 477 for 8+ years with
> very little difficulty. We recently had to leave our tried and true instrument
> behind and bought another one. We've never really gotten the "new" one to
> perform to the standards of our old instrument and I don't know why.
>
> Basically, the problem is one of repetitive yield. The yields look good up to
> about 5 residues and then the data falls off into nothingness (if that's a
> word). We use all of the solvents directly from PE except for R2. We've tried an
> R2 recipe from another ABRFer that's made up of DIEA/H2O/MeOH. PE is trying to
> tell us that we have a bad column and we need to change all of our reagents
> (especially the R2). The column is older, but the separation is still great. I
> actually tried a different column at PE's request, but found it was worse than
> the older one so I switched back. To be fair abour the R2, this is a recent
> switch. We've had inadequate r.y. long before we switched from TMA to the
> homemade solvent. It's also been suggested to us that we're applying the sample
> incorrectly (even though we've done it the same way for 8 years and the problems
> are more recent) and that we shouldn't sequence peptides directly on the resin
> (even though we've done that for 8 years also).
>
> I don't know sequencing well enough to know where to look for the problem. I've
> changed all of the solvents and I don't feel like doing it again without a good
> reason. I really think it's due to something in the cycle optimization, but I
> don't know what to change first (or second...).
>
> I know this is a long message and the solution will probably not be an easy fix,
> but we really need some help.
>
> Thank you in advance for your assistance.
>
> Jeni
>
> Janelle Lauer-Fields
> Department of Chemistry & Biochemistry
> Florida Atlantic University
> 777 Glades Road
> Boca Raton, Florida 33431
> 561-297-2094/Fax 561-297-2759
>