How much do you weigh?

March 13, 2010 12:55 PM

Determining the stoichiometry of large (>4MDa) protein complexes?

I am trying to figure out the best methods to use for determining the stoichiometry/molecular weight of a large protein complex. The minimum the complex will weigh is about 4 megadalton and I am unsure of the maximum (my guess is less than 10 megadalton). I am able to pull the complex out of cell lysate and also may be able to crosslink it.

I am shying away from using mass spec techniques to do this, as the complex includes 60 repeating units of one protein and an unknown number of repeating isoforms of another protein. I feel like the data would quickly become a mess.

Currently I'm thinking about using analytical ultracentrifugtation and/or dynamic light scattering. I am wondering if any has additional suggestions. Also, if you have an opinion on using the two techniques I mentioned (for instance, I am unsure if the weight of my complex is above the detection limits for analytical ultracentrifugation) I would really appreciate it.

posted by sickinthehead to Science & Nature (9 answers total)

This works well:

http://scripts.iucr.org/cgi-bin/paper?S0021889809043076
posted by overhauser at 1:37 PM on March 13, 2010

I think you'd be O.K. with analytical ultracentrifugation and nearly certain you're fine with light scattering techniques (static or dynamic) for a complex of that size.

Some other suggestions for molecular weight determination are size exclusion chromatography and field flow fractionation. However, depending on how accurate of a result you need, you might run into problems if your proteins are highly non-spherical. Both SEC and FFF separate analytes based on hydrodynamic volume, not on absolute molecular weight, and are typically calibrated against spherical standards.

Just for fun, you could also try that old Chem 101 experiment of measuring the osmotic pressure difference across a semi-permeable membrane when you add a known mass of pure protein to one side.
posted by TBAcceptor at 1:56 PM on March 13, 2010

You could make a reference solution of the material in water (known number of grams per liter), and then find out its osmotic gradient with pure water using a semipermeable membrane. This method can produce pretty accurate results (and it is cheap).
posted by candasartan at 3:06 PM on March 13, 2010

I've used gel electrophoresis before to determine molecular weights of heavier proteins. I don't know if something like agarose gel electrophoresis might work for you or if the masses you're dealing with are too high for the technique. I'd also be concerned about complex stability and the limitations of precision in determination of the molecular weight from electrophoresis.
posted by darkstar at 1:59 AM on March 14, 2010

Response by poster: Darkstar: I'm going to use native PAGE just for an initial estimate of mol weight, but I'm concerned about the complex stability with it, too. The equilibrium constant for the complex is unknown and I would imagine if it's not a strong binder that the different mobilities of the species involved could overtake the need for them to stay together.
posted by sickinthehead at 8:09 AM on March 14, 2010

Makes sense. I'm curious about the osmotic pressure test described above. That'd be a pretty elegant way of doing it if it works.
posted by darkstar at 9:54 AM on March 14, 2010

For a huge, somewhat loosely-associated complex, the techniques mentioned above are classic but I urge you to run more than one of them. Huge complexes often dissociate easily, or have poor solubility without detergents (which can bust up the complexes, introducing artifacts). Orthogonal methods give you the best chance of getting the right answer - if they all more or less agree, you have more confidence in your result.

Wow, you actually have access to an analytical ultracentrifuge? I've never even seen one of those beasts in person.
posted by Quietgal at 10:59 AM on March 14, 2010

Response by poster: Quietgal: I am intending to do, at the very least native PAGE, analytical ultracentrifugation and dynamic light scattering right now, as each of these have their own unique caveats of measurement. But that's why I made this post - I'm trying to compile a list of techniques that maybe hadn't occurred to me so that I can see if my original plan is the best or should be modified.

I agree that the osmotic pressure thing would be an elegant way of determining the molecular weight. I am going to have to work out the logistics of that, though.
posted by sickinthehead at 1:18 PM on March 14, 2010

Response by poster: Oh, and I have access to an analytical ultracentrifuge through the core macromolecular interactions facility at my university. Certainly not in my lab (though we do have a number of ultracentrifuges for other purposes).
posted by sickinthehead at 1:19 PM on March 14, 2010