phil at wyatt.com (Philip J. Wyatt) writes:
>We are a small company that manufactures multi-angle laser light
>scattering detectors (MALLS) used in combination with chromatographically
>separated samples to determine their absolute molecular weights and sizes
>and the distributions of these quantities.
> 1) What do you think of the chromatographic separation process as
>a tool for characterizing protein samples (assuming that an absolute
>determination may result)?
>===============
The only technique that seems to yield definitive results is size-exclusion
gel filtration. By definitive I mean results you can talk about results as
reasonably solid evidence that there is an aggregation difference. Even in
the absence of exact stoichiometries (i.e. eluted mol. wt./subunit mol.
wt.) stating that there is a difference is reasonable.
I have one protein under study that seems to elute from ion exchange resins
as a split peak. Subject the eluted samples to a molecular weight sizing
gel and both elute at the same point. Re-elute these samples from the mol.
wt. sizing gel on ion exchange and both now elute at the same salt
concentration. I believe the separation afforded by the first ion exchange
column is real, but cannot definitively determine "why" the protein split
on the column. I suspect an aggregation phenomenon -- something an on-line
mol. wt. detector could determine (we looked at these several years ago and
they couldn't correctly assess the mol. wt. of a test protein we had --
might be time to see if the years have been good to the technique -- hmmm,
send me some literature. :-)
> 2) If you use such techniques, what type do you use? (E. g.
>reverse phase, size exclusion, ion exchange, capillary electrophoresis)
Of your list, size exclusion and ion exchange are the most popular (at
least with our lab.) Reverse phase and capillary electrophoresis
techniques almost always use conditions that are far too harsh for most
proteins. It would be foolish to try and assess aggregation state of a
protein after it had been subjected to trifluoroacetic acid, phosphoric
acid, organic solvents, etc. Reverse phase and capillary seem suited to
purifying a polypeptide chain (as opposed to purifying a native protein.)
> 3) Do you think the buffer and/or the mobile phase can (will)
>affect the composition of your sample? (E. g. might you detect
>aggregates that were not present in your sample? Could some of the sample
>stick to the columns during the separation process itself?)
I would say yes. With two proteins we currently work with, aggregation,
whether part of the native "operational mode" of the protein, or caused by
buffer conditions is observed (one proteins aggregation state depends on
the presence or absence of one substrate.)
> 4) If a column structure could be developed that would permit
>the use of any type of mobile phase without affecting the separation
>process itself (i. e. no sticking and no false aggregation), would that
>make such chromatographic techniques more attractive? (We do NOT
>manufacture columns!)
This would be a neat trick if you could do it. Your description pretty
much describes the goal of a mol. wt. sizing column. A priori, this would
not seem to apply to the ion exchange, reverse phase, affinity
chromatography, capillary, etc..
It sounds like your company is focussing on devices for on-line
identification of mol.wt. from chromatographic resin (be they ion exchange,
mol. wt. sizing, or whatever.) This is useful. However, many of the
aforementioned chromatographic techniques are being used in the hopes that
differential aggregation (in our case it would protein-protein interactions
between a regulatory protein and its substrate) can be analyzed. Of just
as much utility would be direct measurement of the mol. wt. distribution
within a single (stirred?) vessel versus time (i.e. add substrate protein,
check mol. wt., add regulatory protein, check mol. wt.) Evidence of
changes, even if not definitive in terms of mol. wt., would be very useful.
--
Eric Larson | University of Illinois at Urbana-Champaign
USDA/Agronomy | 190 PABL; 1201 W. Gregory; Urbana, IL 61801
elarson at ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419
Fidonet: 1:233/4.1 | My opinions are my own, but correct :-)
