RE: In-gel AND on-membrane digests
Joseph Fernandez (fernaj@rockvax.rockefeller.edu)
Thu, 5 Mar 1998 08:58:58 -0500 (EST)
Axel, Ken and others,
The limitations for obtaining internal sequence information I
believe are more from the HPLC's, sequencer's and mass specs than the
actual digestion techniques (at least for PVDF). For standard proteins in
one lane, we are able to get GOOD peptide maps and obtain internal sequence
data from at least 2 pmol of material (50 - 75K) loaded onto a gel and
transferred to PVDF (higher binding version). We see clear peptides on a
C18 VYDAC 1mm column using a 1090 HPLC. We have been able to get sequence
data from these peptides with initial yields of 0.3 -0.8 pmol (15-40%), all
on our Procise cLC sequencer. Although we have not run these on our
Procise HT instrument I would predict only the peptides with yields greater
than 0.5 pmol on the cLC would have produced data. Lower MW proteins we
tend to need about 5 pmol to get satisfactory data. Also based on staining
intensities we believe we have analyzed many real samples in the 1-5 pmol
range. We have also analyzed many sample in-gel in this range with equal
success.
> The choice between nitrocellulose, PVDF or in-gel are more personal
>preference and dependent on data needed. You cannot put a gel or
>nitrocellulose in a sequencer and get direct N-terminal data as with PVDF;
>however, if an investigator has very low amounts of starting material and
>only wants internal data then the recommendation would be to go with
>in-gel digestion so as to eliminate the chance of sample loss during
>transfering to a membrane. Although nitrocellulose can also produce good
>recovery, we do not typically analyze these samples preferring PVDF or
>in-gels for the above reasons.
>>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 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
>
>
>
Joseph Fernandez
Associate Director
The Rockefeller university
Protein/DNA Technology Center
1230 York Ave. New York, NY 10021
Phone: (212)-327-8869
FAX : (212)-327-8620
email: fernaj@rockvax.rockefeller.edu
Lab Web Page: http:\\pdtc.rockefeller.edu