I do not want to throw oil in the fire, but the problem with this study lies
precisely in the protein amounts that have been analyzed. With 50 pmol on hand,
and
having good sequencers and/or a good LC/MS system (or, of course, a MALDI-Tof),
I am
quite confident to get a number of relevant amino acid sequences, idependently i
f I
get the protein in a gel, on a PVDF membrane or on a nitrocellulose membrane
because, even if the recovery yields are low, there is still sufficiently materi
al
available to perform the analyses (20% of 50 pmol is still 10 pmol, a lot for mo
dern
MS instruments or sequencers). Now, what I would like to see is the same study d
one
with only 5 or 1 pmol of protein available, because now matrixes begin to
substantially interfere with the analysis. Personally (and, please, do not see t
his
comment as an attack on your preferred method; this is just a matter of personal
preference, as Ken mentioned), I have not managed to get substantial good result
s
with PVDF membranes. Although I used to prefer nitrocellulose membranes over in
-gel
digest, I also had to realize that recovery yields substantially decrease once t
he
low pmol is reached. I do not like present in-gel digest protocol because you h
ave
to clean up the digests from a lot of garbage before applying to a MALDI target
or a
capillary column. And clean-up, at least in my eyes, means losses.
So, what I am heading to is the following. I do not think that the current bottl
e
neck is the instrument sensitivity (although this could be discussed for Edman
sequencers). Most of modern MS instruments have currently sensitivity going to t
he
low fmol and heading to the amol. However, there are not many publications deal
ing
with the sequencing of novel protein below the pmol. So where is the problem?
Consistently, one of the problem that I have observed here in my lab and at othe
rs
is sample handling. We have to realize that methods for protein digestion have n
ot
changed dramatically over the last 5 years. Yes, of course, we have miniaturized
the
equipment, tried to worked with smaller volumes, concentrated the proteins in
smaller areas. However, there is no true consensus on how it should be done. The
ABRF could take a lead here on conducting a study on how to manipulate very low
amounts of proteins in a real world fashion, that is a protein, let's say, that
have
been isolated from a biological tissue using conventional purification technique
and
contaminated with the usual cocktail of salt and detergents that typically dooms
my
own analysis. Because if we have currently techniques to retrieve 20% of the
available protein amount, we got to find something to tap in these remaining 80%
!
Kenneth Williams wrote:
> "Finally, the answer to the question of which approach (in-gel or on P
VDF
> digestion) is best to take for internal protein sequencing is that the two
> methods appear to be quite comparable. That is, when peptide recoveries
> are calculated from the Edman sequencing yields divided by the amount of
> protein in the stained gel or PVDF band (as opposed to the amount that was
> loaded onto the gel), the target peptide recoveries for both PVDF and gel
> samples were just above 20% in the 1997 study (Table 3). The optimal
> method for a specific project depends upon the protein of interest and the
> personal preferences and level of expertise that the core lab to whom the
> sample will be submitted has with each of these approaches. Based on two
> ABRF studies, each of which involved 40 core laboratories, we conclude
> that if a 50 pmol protein sample is subjected to SDS PAGE and then prepared
> according to the protocol recommended by the core laboratory to which the
> sample will be submitted, the probability of obtaining internal peptide
> sequences is likely to be much higher than 83%."
>
> Ken Williams
-- --------------------------------------------------------------------------------
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 Canada
tel. + (514) 428-3428 fax + (514) 428-4900