While I would also like to put my hand on a BrCN protocol for the digestion of
low amounts of proteins, please let me present you with an alternate strategy
for determining the N and C termini of a known protein.
The main argument against an usual peptide mapping strategy for the
determination of N/C termini of protein is that it is usually (not always)
difficult to determine unambiguously which peptide (or most likely which
peptideS) are the true N/C termini. Let's simply assume that your known protein
has ragged termini: during a digest, the several N/C terminal peptides may end
up to be "lost" in the maze of the other peptides that you generate. Also, if
the mass of a N/C terminal peptide does not fit with a calculated mass, this
becomes quite complicated as you will then have to generate tandem MS data to
confirm the sequence, a task that may or may not work well on a MALDI-ToF.
Having written that, what are the alternatives? The strategies presented below,
ideally, aim at specifically enriching for the peptides that are N/C terminal
while throwing the rest out.So:
1) N-terminus: while 1 pmol may not represent a lot of protein, this should be
still sufficient for the good old Edman chemistry (assuming, of course, an
unblocked N-terminus)with an optimized sequencer. In your area, ask for Paul
Tempst at Sloan Kettering, probably one of the most experienced person in this
area. Because you want to determine a N-terminus, and not a long stretch of
amino acids, and because you are dealing with a known protein, even dubious
cycles which would be ignored in a normal case can probably still be
interpreted. Although the method is now more than 20 years old, Edman Chemistry
still remains the most unambigous method to determine an amino acid sequence to
date. Alternatively, if you suspect a blocked N-terminus, you can use the
strategy developed by Akiyama et al. (1994) Anal. Biochem. 222:210-216, to
selectively isolate the N-terminal peptide from a digest. The protein is first
treated with succinic acid anhydride or maleic acid anhydride to block free
amino groups and digested with chymotrypsin. When the digest is passed through
an activated BrCN sepharose column, all peptides containing a free NH2 groups
will be retained whereas the succinylated or maleiated N-terminal peptide will
pass through. The very much simplified mixture can now be analyzed by the method
of your choice, for example, by MALDI-TOF.
2) C-terminus: The determination of C-terminal peptides is slightlty more
complicated since 1 pmol starting material is hopelessly below the sensitivity
of current C-terminal sequencers. Here again, the trick is to enrich for the
C-terminal peptide(s) after cleavage with the appropriate endoproteinase. A
strategy described by Kumazaki et al. (1987) J. Biochem. 102:1539-1546, is using
trypsin to cleave the protein and to capture newly generated tryptic peptide
(which should all have a C-terminal K/R residue, EXCEPT the C-terminal peptide)
on an immobilized anhydrotrypsin affinity resin. The flow-trough should at least
in principle contain the C-terminal peptide(s) that can be analyzed by the
method of choice. This strategy has of course a caveat: if the protein ends up
with a K/R, not likely to be cleaved by trypsin in this condition, the
C-terminal peptide us then unlikely to be retrieved.
Having written that, I am well aware that all these methods have not be tested
for protein that are below the pmol level (although the procedure developed by
Akiyama has been tested at the 10 pmol level by people at Perseptive
Biosystems). I have no feeling what so ever what the losses are going to be
using this affinity columns. However, using a calibrated protein as standard,
you should be able to determine rapidly whether this strategy will work for you
or not. In any case, I wish you good luck (you will need it!) in this project
Axel Ducret
James Farmar wrote:
> Dear ABRF Colleagues,
> Does anyone know of a tried and true method/protocol using cyanogen bromide
> to digest a low level amount (<1 pmol) of a protein in a gel slice or a
> pvdf band? The method should be amenable to extraction of the peptides and
> MALDI analysis after the reaction. Additionally, I would prefer a method
> that avoided formic acid so as to avoid formylation of the cleavage products.
> I want to determine the N and C termini of this known protein. The
> resulting peptides will have a MW between 1000-6000 g/mole. I'm willing to
> give the digest a try but the researchers have to go through a considerable
> effort to get the protein.
> My thoughts:
> pvdf: CNBr/0.1M HCl/ACN or MeOH (for wetting?); extraction as per normal
> digest
> gel: will CNBr leave the polyacrylamide alone?
>
> Thanks!
>
> Dr. Jim Farmar
> MicroChemistry
> L.F. Kimball Research Institute
> New York Blood Center
> 212 570-3128
> 212 570-3195
-- Axel Ducret, Ph.D. Senior Research Biologist Merck-Frosst Canada Inc. Dept. Biochemistry and Molecular Biology P.O. Box 1005 Pointe-Claire-Dorval PQ H9R 4P8 Canadatel. + (514) 428-3428 fax + (514) 428-4900