Re: Digest/ProtSeq
Joseph Fernandez (fernaj@rockvax.rockefeller.edu)
Tue, 3 Mar 1998 08:12:30 -0500 (EST)
>
>To: "Rebecca Ettling" <rebecca_ettling@smtpgw.musc.edu>
>From: Joseph Fernandez <fernaj@rockvax.rockefeller.edu>
>Subject: Re: Digest/ProtSeq
>Cc:
>Bcc:
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>
>Rebecca,
> We have been performing in-situ digestions on PVDF for internal
>sequence analysis successfully for about eight years now (see Fernandez et
>al 1992, Anal Biochem 201, 255-264 and Fernandez et al 1994, Anal Biochem
>218, 112-117). Peptide recoveries have always been good, with yields
>comparable to in-gel digests (providing you get good transfer to PVDF). If
>I recall the ABRF internal sequence committee found good results from PVDF
>samples in their past studies. In addition we have been able to use
>several enzymes such as trypsin, endoproteinase lys-C, endoproteinase
>glu-c, clostripain (arg-c), endoproteinase asp-n, and thermolysine. Here
>are some suggestions
>
>1) Are you adding nonionic deterrgents to your digestion buffer? You need
>to use something like reduced triton X-100, tween-20, or more recently
>zwitterionic detergent. All appear to produce equal recoveries in our
>hands. Otherwise the yields are poor.
>2) The reduction alkylation should not be a problem on enzyme activity
>unless you are using ecessive amonts. We usually use 5 ul of 45 mM DTT
>followed by 5 ul 100 mM iodoacetamide.
>3) Are you digesting the band exact band you tried to sequence or are you
>digesting an "unsequenced" band? If it right out of the sequencer then
>the lysines are probably modified with PITC and the harsh Edman chemistry
>makes peptide recovery (via enzymatic techniques) impossible. We have
>tried many times always with negative results. If you have previously
>sequenced the band then perhaps a chemical procedure described by Dan
>Crimmins would be more appropriate.
>4) The cappillary system (ABI's?) may not be too compatable with some of
>the high detergents/salt soultions. If you are using nonionic detergents
>are you what volume are you using, are you performing extractions, and
>what volume are you injecting? I know on our system the detergent elutes
>later and you can observe it wetting the PVDF (again on the ABI system) in
>an odd fashion, sort of just layering. ANother thing is we found the
>reduction/alkylation buffer results in very high background in the
>beginning of cappilarry runs. Perhaps you can try performing the R/A in
>100 mM tris after wetting the PVDF with methanol; discard the R/A buffer
>and add the detergent containing buffer with enzyme.
>5) As stated by others previously, heavily glycosylated proteins are
>difficult to digest on PVDF (In-gel people, is this true for in-gel
>digests also?). We have also run into problems in getting data from
>darkly stained amido black bands that are highly glycosylated and usually
>suggest investigators try to remove the sugars prior to resubmitting the
>sample. Protiens that won't digest (we have seen maybe three in 8 years)
>are rare but they do occur.
>6) Did the investigator use Coomassie Blue R-250 or G-250. Our experience
>is that the R-250 is very dirty and produces enough artifacts to make
>peptide identification difficult. G-250 is cleaner and more applicable to
>peptide mapping. Is the blank digestion very high where the contaminant
>peaks interfere with peptide identification during the ample run?
>
> Hope I have been able to help.
>
>Joe
>
>>Hi Everybody,
>>
>>My problem:
>>
>>A customer brought me a Coo-blue stained blot with a faint but visible
>>band on
>>it to sequence. I put it on my sequncer and got no sequence. I told the
>>customer that either there was less protein in the band than we thought or it
>>was N-terminally blocked.
>>
>>We sequenced again with (she tells me) more sample -- still no sequence. We
>>decided that it must be blocked and decided to digest it.
>>
>>The expected protein has plenty of tryptic sites so we red/alk and then did a
>>tryptic digest(on PVDF). (DTT/Iodoacetate, Promega trypsin) I ran the
>>digest
>>on a sample slice and a blank slice (of PVDF) in parallel. When I ran the
>>digests out on my microcapillary HPLC, the digest of the sample PVDF looked
>>exactly the same as the digest of the blank lane. I have to conclude
>>that the
>>protein didn't digest, but why ?
>>
>>At this point my customer only has a little bit of the sample left (we only
>>get 1 more shot). She needs enough sequence to prove that the protein is
>>what
>>her western's have led her to believe it is.
>>
>>My questions:
>>
>>1) Are there proteins out there that are Trypsin resistant even after
>>reduction/alkylation ?
>>
>>2) What other possible explanations are there ?
>>
>>3) What would you do next ? (Our only idea is to try CNBr?)
>>
>>Thank you all for all the past help and thanks in advance for help with this
>>problem.
>>
>>Weather Inconsequential(Charleston S.C.):
>>Spring has sprung and if you can avoid sinking up to your ankles in mud, the
>>azaelas are beautiful !
>>
>>Rebecca P. Ettling
>>Biotechnology Resource Laboratory
>>Protein Sequencing and Peptide Synthesis Facility
>>Department of Biochemistry (Rm 505)
>>Medical University of South Carolina
>>171 Ashley Avenue,
>>Charleston, SC 29403
>>Tel.: (803) 792-1271
>>Fax: (803) 792-1264
>
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