Dear Ricardo,
Have you run a blank reaction mixture on the HPLC? When I was
cleaving proteins with iodosobenzoic acid and separating the fragments by RP
HPLC, I ran several blank reaction mixtures containing 80% acetic acid, 4M
guanidine HCl, o-iodosobenzoic acid with and without p-cresol. I always saw
two very large, very annoying, late-eluting peaks which were not present
when only acetic acid and guanidine HCl were injected. I was never able to
effectively extract away whatever was causing these peaks. This is probably
why I switched to using BNPS-skatole for cleavage at tryptophan: those
reaction products were easily extractable by ether.
My point is that your lack of sequence and MS data may be due to the
fact that the peaks you collected did not contain any peptide.
Hope this helps.
Carol
At 09:53 AM 8/24/00 -0300, you wrote:
>I am facing a problem and I need some help to solve it. I cleaved a long
>peptide with iodosobenzoic acid and I separated the peaks by reversed-phase
>HPLC. They were big peaks, many surpassed the scale of 1.0 AUFS. After
>drying in SpeedVac, I applied some peaks in mass spectrometer and automated
>sequencer. I didn't obtain much sign. Since the peaks went out with a
>relatively high concentration of acetonitrile (>50%), they should be enough
>hydrophobic. Because that, I suppose they should be stuck to the walls of
>the polypropylene microcentrifuge tubes. In order to solubilize them, I
>intend to use a 3:1 solution of methanol:chloroform. Then I intend to add
>water and acetic acid in order to apply into an electrospray mass
>spectrometer. Could anybody tell me if the chloroform interferes in the
>electrospray process? Some other suggestion for the material loss?
>
>Thanks in advance.
>Prof. Ricardo B. Cunha
>Brazilian Center for Protein Research and Services - CBSP
>Laboratory of Protein Chemistry and Biochemistry
>Institute of Chemistry
>University of Brasilia
>Brasilia - DF Brazil
>ZIP CODE: 70910-900
>
>Tel: 55-21-61-307-2142
>Fax: 55-21-61-272-4548
>mailto:rbcunha@unb.br
>http://www.geocities.com/Athens/Acropolis/5221
>http://www.unb.br/cbsp
Carol Manley Beach, Ph.D.
Protein Sequence Analysis
Macromolecular Structure Analysis Facility
232 Combs Cancer Research Building
University of Kentucky
Lexington, KY 40536-0096
cmbeach@pop.uky.edu
NOTE NEW AREA CODE: Mandatory after Oct 1, 2000
(859)257-4932 (office)
(859)257-4475 (lab)
(859)323-3909 (FAX)
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