Just my 2 (canadian) cents worth of comments to Amos' letter quoted
below.
The salt concentration in in-gel digest preparation does not need to be
a problem. We and others have suggested that, actually, trypsin works
fairly happily in low-salt buffers (provided the pH is about 8.0). Thus,
Hanno Langen suggested at several meetings (and in one paper) the use of
2 mM Tris pH 8.0 as a digestion buffer (which can be used "as is" for
MALDI-ToF analysis) while I have used for several years now a 15 mM
N-ethylmorpholine, 5 mM acetic acid buffer (no salt) for doing all my
in-gel digest with no apparent difference compared to a typical
ammoniumbicarbonate digest.
Keep the good ideas coming! I have also been struggling with these
phosphopeptides and any suggestion is step in the right direction
(although the road may encompass a couple of turns ...)
Axel
Derek Bradley wrote:
> Amos,
>
> For the uninitiated among us (especially me!!) can you give a brief
> description of HILIC?
>
> Thanks,
>
> Derek Bradley
> Dept. of Medicine
> UCL
> London
>
> At 12:22 22/02/01 Thursday, Amos Heckendorf wrote:
>
>> Len
>>
>> Thank you for the posting. It is always entertaining to have a
>> major marketing company admit some limits. Do you think that they
>> will now come out with a product for crow, having acquired a good
>> taste of it here?
>>
>> Dr. Alpert and I have posted several times on the use of HILIC for
>> detergent removal, as well as for phosphopeptide retention, rather
>> than the IMAC approach. However, there has not been much feedback
>> on this subject. I am curious as to whether anyone has had success
>> with this? (One underutilized function of this discussion group
>> would be to allow more manufacturer postings so that the free market
>> of ideas would more quickly move us onto better products or
>> techniques. We have to agree to be civil about it though!)
>>
>> Part of the problem with the HILIC approach is that the salt
>> concentrations are somewhat high in in-gel digests and one needs to
>> carefully manage this aspect of the problem so that the polar
>> peptides will actually stick. Keeping the molarity below 50mM is
>> only a benchmark level. It may require even less salt present, so a
>> C18 step is not out of the question here, but the salt tolerance of
>> HILIC methods needs to be better defined.
>>
>> Additionally the capacity of ZipTips or their competitors (PrepTips
>> and SuproTips) are somewhat low as compared to MiniSpin columns with
>> HILIC chemistries. The liquid volumes of spin columns are not
>> substantially larger than the coated wall or plugged flow tips, but
>> the bed volumes are much greater. This means that the amount of
>> polar solute retained could be greater, and you would not need as
>> clean a sample to be successful at this.
>>
>> We have samples of both types of products available for those of you
>> interested in framing this problem in more realistic terms other
>> than the standards that have been run. Please let me know if you
>> are interested.
>>
>> I hope to see many of you at San Diego where we can discuss this
>> further.
>>
>> Sincerely,
>> Amos Heckendorf (amos@nestgrp.com)
>> 508.481.6223
>>
>> ----------
>
-- ------------------------------------------------------------------------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
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