delta-pI of single phosphate incorp into peptide?

Bob Steinberg rsteinbe at etowah.ouhsc.edu
Fri Feb 27 20:34:28 EST 1998



Darren Tyson wrote:

> Greetings,
>
> I'm trying to analyze phosphorylated proteolytic peptide fragments by
> isoelectric focusing.  My question is what would the change in pI be
> if a single phosphate was incorporated?  Would it depend on the rest
> of the peptide sequence?  (I can't remember how pI is calculated.)
>
> Mainly I want to be able to distinguish among one, two, three, etc.
> phosphates incorporated into a single peptide fragment.  Is this
> possible by IEF or do I need to sequence radiolabeled fragments
> looking for 32P incorporation onto individual residues?
>
> Any help would be greatly appreciated?
>
> Cheers,
>
> Darren
>
> --
> Darren Tyson, Ph.D. Candidate
> Cell and Molecular Biology Program
> Saint Louis University, St. Louis, MO
> tysondr at nospam.slu.edu
> (remove nospam. before mailing)
> http://users.primary.net/~darren/

I don't know about the change in pI, but I do know that you can detect
the effect of single phosphate additions by IEF-- we've used O'Farrell
2-D gels routinely to monitor phosphorylation changes in intact
proteins-- as you would expect, the magnitude of the shift is larger the
smaller the protein-- if you have lysines on your peptides, you can
calibrate your IEF gels for single charge shifts by carbamylating (easily
accomplished by heating samples in urea [without any ampholines or other
sources of amino groups that might scavenge the isocyanate formed by urea
decomposition]).I hope this helps.

Bob Steinberg






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