I recall problems occasionally with COMPLETE blockage of the amino-termini
of CNBr-cleavage products. We traced the problem to a particular bottle of
formic acid. This was pre-mass spec, so we had no idea whether the lysines
were modified too. As someone once wryly commented, "the person who bought
that bottle of formic acid is no longer with us!".
Ken
Kenneth A. Walsh
E.W.Davie/ZymoGenetics Chair of Biochemistry
Box # 357350
University of Washington
Seattle WA 98195
walsh@u.washington.edu
Phone 206-543-1768
FAX 206-685-9231
On Fri, 31 Jul 1998, Mark Lively wrote:
> Fellow protein chemists,
>
> We have produced a recombinant protein of approximately 16,000 Da as a fusion protein with maltose binding protein. The desired protein is basic with 15 Lys and has no Met's. We have used CNBr cleavage in formic acid to cleave the fusion protein and then isolated the desired 16 k product by reverse phase hplc. The purified product was identified by amino acid sequence analysis.
>
> Electrospray ionization ms of the purified product yields a family of peaks starting very close to the target mass but increasing in mass by approximately 28 Da per "adduct". The actual m/z values increase by 21.88, 27.75, 29.0, 27.5, 26.2, & 25. The principal ion is at approximately the expected mass plus 4 X 28 Da.
>
> QUESTION: Are we observing extensive formylation of our protein and, if so, have others seen this result with other CNBr digests? Or is there another explanation for the formation of these different +28 Da adducts?
>
> Thanks in advance for your help.
> Mark Lively
>
>