Is it possible that your enzyme is glycosylated? Variable sialylation
would potentially give rise to multiple IEF bands. Also, it is possible
that C-terminal processing (exoproteolytic truncation) would remove a
charged group, introducing yet further IEF heterogeneity, and yet the
peptide mapping procedure might not be optimized to separate the
truncated peptide from its native counterpart. You might be able to
address both of these potential problems at once by performing LC-MS
peptide mapping in order to assign all of the peaks to enzyme fragments
(and potential modifications), including its performance in parallel on
native and deglycosylated enzyme if it is glycosylated.
Mike Klein
Amgen, Inc.
> ----------
> From: Jason Eglinton[SMTP:jeglint2@waite.adelaide.edu.au]
> Sent: Monday, February 16, 1998 3:53 PM
> To: Recipients of ABRF List
> Subject: charge heterogeneity
>
>
> I have a problem understanding the source of charge heterogeneity in
> an
> enzyme I work with. The preparation is pure as judged by complete
> peptide
> mapping, but exhibits multiple (5 or 6) bands on a native IEF gel. The
>
> sample is reduced prior to IEF, so it is not due to dimer/aggregate
> formation. The enzyme has proven resistant to ionisation, so ESI and
> MALDI MS have not helped.
>
> Does anyone have examples of charge heterogeneity due to
> conformational
> changes or interaction with carrier ampholytes? I look forward to your
>
> suggestions.
>
>
> Jason Eglinton ph (08) 8303 7286 fax (08) 8303 7109
> Department of Plant Science
> Waite Agricultural Research Institute
> The University of Adelaide
>
>