FW: From maria Lorenzi ( Italy )
Wilhelm, Randy R (Randy.Wilhelm@mkg.com)
Thu, 5 Nov 1998 11:18:47 -0600
> ----------
> From: Wilhelm, Randy R
> Sent: Wednesday, November 04, 1998 11:29 AM
> To: 'Maria Lorenzi'
> Subject: RE: From maria Lorenzi ( Italy )
>
> On Nov 4th Maria wrote:
>
> "And please, could you suggest some procedures to see the tryptofan?
> Ciao ciao
> Maria"
>
> My experience is mainly in mass spectrometric techniques not automated
> sequencers, so I have a suggestion using MS. If the Trp is near the
> C-terminus and this is a C-terminal acid, you could do a carboxypeptidase
> Y (CPY) partial digestion of the material and get some ladder sequence
> information from the C-terminus. Analysis can be by MALDI or ESI-MS.
> Time dependent digestion can be performed, with the digestion increasing
> through each subsequent amino acid moving toward the N-terminus.
>
> A reference for this procedure, although it is fairly straightforward is
> below:
>
> Anal. Chem. 1995, 67, 3971-3978
>
> CPY is available from Sigma Chemical (cat. no C3888)
>
> Randy Wilhelm
> Sr. Research Chemist
> Mallinckrodt, Inc
> Discovery Research - Peptide Team
> rrwilhe@mkg.com
> (314)654-3965
>
>
>
>
> ----------
> From: Maria Lorenzi[SMTP:mlorenzi@ascu.unian.it]
> Sent: Wednesday, November 04, 1998 5:55 AM
> To: Recipients of ABRF List
> Subject: From maria Lorenzi ( Italy )
>
> Hi, I'm Maria.
> I need help. Yesterday I load my toxin in the Sequencer to obtain the
> total
> sequence of the monomer. We obtained it even if it is difficult to say if
> the monomer has 50 residues or 51; in fact the background at the end is
> high
> and we don't see an increment of any aminoacid.
> Anyway, the 49 residue is a Tryptofan and in this sequencer there is the
> problem that we cannot see it. I ask you why and in what way we can see
> it.
> In this sequence we see, in that cycle, the increment of two peaks that
> decrement in the subsequent cycle but we don't see the tryptofan. One of
> these peaks is between the E and H while the other is immediately after
> the
> alanine.
> An other person had this problem with his sequence, he sequenced his
> peptide, he got high sequence but in the corrispondence ot the tryptofan (
> he previously knew that there was a tryptofan from the gene )he didn't see
> anything but the increment of a peak closed the prolin.
> It is strange that it could be a non proteic amino acid because the
> other
> previous aminoacids are proteic.
> Could you give an answer about these questions. In fact we want the total
> and sure sequence of the toxin to clone the gene.
> I thank you and I wait your anwser.
> And please, could you suggest some procedures to see the tryptofan?
> Ciao ciao
> Maria
>
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