ProtSeq

Deb McMillen (mcmillen@morel.uoregon.edu)
Tue, 11 May 1999 18:27:17 -0700 (PDT)

Hi, all,
I'm sequencing a protein expressed in E. coli and am definitely seeing an
N-terminus that was created by cleavage at an FS bond--I have this on my
list of acid labile bonds, but how common is this? It is a very large
percentage of the prep. And does this cleavage happen primarily because
of the neat TFA delivery in the reaction cartridge--or does this require
water for the cleavage and happen before the sample has hit the protein
sequencer? I loaded the sample out of Tris onto a ProSorb--and did use
50ul of 5% acetonitrile 0.1% TFA after wetting with methanol to "wet the
ProSorb" (I like to make sure that the ProSorb-filter combination is
really working before I throw the protein on it) and then put through 400
ul of the Tris (I don't know their pH) and then wash with 2-100 ul
aliquots of 5% acetonitrile 0.1% TFA, partially dry the ProSorb, then wet
with 10ul of methanol and dry under a stream of nitrogen and then pump
under hi vac before going to the sequencer.

We are going to check the mass of this protein (mixture) by MALDI--I'd
like to know if the prep arrived like this--And I thought I'd prepare a
second ProSorb prepared without any TFA.

While I'm on this topic of acid and base labile bonds, which of
the following is most labile (I have always understood DP to be the most
sensitive)

Acid labile DP DG GP ST and FS

Base labile TP RP NG

I also understand that some bonds are labile under either acid or base
conditions.

Is there a good reference out there that I can look up?

Thanks for your help,
Deb McMillen
Institute of MOlecular Biology
University of ORegon
EUGene OR 97403