Thank you so much for posting your detailed comments (copy below) about =
in
gel/PVDF digests and please by all means continue to throw all the "oil i=
n
the fire" that you like - it is the only way to shed some light on the
problems.
=09
We agree that 50 pmol is a rather large amount - that frankly we often d=
o
not see. The minimum amount that we believe is generally recommended (by
laboratories that specialize in this area) for conventional Edman interna=
l
sequencing is 5-10 pmol with less being needed for MS-based protein
identification. However, in setting up our studies we have to take into
account the varying degree of expertise of our members - for instance, we
were delighted that several participants in our current study used our
sample to try in gel digests for the first time. So, we tried to strike =
a
balance by including a 10 pmol minor component (along with the 50 pmol
major protein) in our current study. We hope you will find the results o=
f
this study, which will be presented at the ABRF Meeting in a few weeks, o=
f
interest.
With regard to sample clean-up following in gel digests, in our opinion=
,
it is not generally needed when there is sufficient protein to permit
MALDI-MS to be carried out on <10% aliquots of in gel digests of single,
Coomassie blue stained gel bands that are carried out as described at the
references that are given below. Approximately 150 in gel digests have
recently been directly subjected to MALDI-MS under these conditions
following a protocol:
=20
http://info.med.yale.edu/wmkeck/geldig3.htm)=20
that is very similar to the representative protocol on the ABRF Internal
Sequence Research Group Web Site:
http://www.medstv.unimelb.edu.au/ABRF/ResearchCommittees/intprotseqrescom=
m.h
tml
In each case the MALDI-MS has been carried out on a microliter or so of t=
he
digest and the remaining gel/digest then frozen (as is) while the MALDI-M=
S
is carried out and the database searching completed. Of these 150
user-submitted, "unknown" samples, 65% have been identified by peptide
mass searching via the ProFound algorithm. Please note these samples wer=
e
not "extracted" and that the fraction of proteins identified increases to
above 90% in the case of an organism like yeast whose genome has been
sequenced.=20
Sample clean-up or repeated cycles of vacuum centrifugation (to lower th=
e
ammonium bicarbonate level) may well be needed at extremely low (<pmol)
levels and when larger fractions of the digest are subjected to MALDI and
other types of MS. =20
A number of laboratories have developed methods to clean-up low pmol
levels of sample digests using reverse-phase material either self-packed =
or
in a small column. A few comments on these approaches from members of ou=
r
committee are listed below.=20
One of our members (Ulf) notes sample clean-up takes less than 15 minute=
s
(for a beginner) on a one-time use "pico-column", made from an Eppendorf
"Geloader" tip, into which is packed about 1 =B5l of Poros coarse particl=
es.
After loading, washing and eluting the peptide mix in a few microliters,
MALDI-MS can then be carried out "undisturbed". At this level the
non-desalted material does not provide a useable MALDI spectrum. =20
Another of our members (Roland) adds, "We only use the cleanup for the
very lowest level of samples, probably on the order 50 ng or less. Then =
we
commit approx 25-33% of the sample to clean up, reserving the rest for
nanoES. We also find we need to use the columns as concentrators when we
had to use large digestion volumes because the spot was from a prep gel.
One extra trick which I picked up from Peter Roepstroff's lab is to crimp
the pipet tip by bending it in half before loading the Poros . This
effectively traps the beads and you no longer have to take pains to keep =
it
from going dry. This tremendously simplifies the elution step."
Scott adds, we have carried out a detailed comparison (Electrophoresis
(18, 369-381, 1997), of in-gel and on-membrane digests using standard
proteins and the results obtained indicated the sample clean-up using a
reverse-phase method (e.g. BioTechniques 22:244-250, 1997) is required fo=
r
successful MALDI-MS peptide mass-mapping (or PSD-MALDI-MS) of low to
sub-pmol levels of in-gel digested samples.
Other relevant references are:
=20
Lui et al. (1996) Anal Biochem. 241, 156-166.
Kussmann et. al. (1997) J. Mass Spectrom. 32, 593-601.
Shevchenko et al., (1996) Anal. Chem. 68, 850-858
Otto et al, (1996) Electrophoresis 17, 1643-1650
Your remaining points about the need to improve methodologies, the very
few laboratories that carry out sequencing of less than pmol amounts of
novel proteins and what happens to the "missing" 80% of an average digest
are extremely well taken and map out some very challenging areas for futu=
re
studies.
Thanks again for taking the time to post your comments,
Roland Annan, Ulf Hellman, Scott Patterson, Kathy Stone, Kristine Swider=
ek
& Ken Williams
_________________
(Copy of 3/4 posting by Axel Ducret)
Ken,
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 pm=
ol
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, independently if I get the protein in a gel, on a
PVDF membrane or on a nitrocellulose membrane because, even if the recove=
ry
yields are low, there is still sufficiently material available to perform
the analyses (20% of 50 pmol is still 10 pmol, a lot for modern MS
instruments or sequencers). Now, what I would like to see is the same stu=
dy
done with only 5 or 1 pmol of protein available, because now matrixes beg=
in
to substantially interfere with the analysis. Personally (and, please, do
not see this comment as an attack on your preferred method; this is just =
a
matter of personal preference, as Ken mentioned), I have not managed to g=
et
substantial good results 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 the low pmol is reached. I d=
o
not like present in-gel digest protocol because you have 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, wh=
at
I am heading to is the following. I do not think that the current
bottleneck is the instrument sensitivity (although this could be discusse=
d
for Edman sequencers). Most of modern MS instruments have currently
sensitivity going to the low fmol and heading to the amol. However, there
are not many publications dealing 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 others is sample handling. We
have to realize that methods for protein digestion have not 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 th=
e
usual cocktail of salt and detergents that typically dooms my own analysi=
s.
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%!