29 March 2010

Native Mass Spectrometry

We’ve recently blogged on the biophysical techniques surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). This post discusses native mass spectrometry (MS), which was reviewed in Future Med Chem earlier this year. (Thanks to author Denis Zeyer for pointing this out).

Mass spectrometry involves ionizing a molecule, measuring its mass-to-charge ratio, and using that ratio to determine molecular weight. Since the process occurs in a vacuum under high electric fields, biomolecules such as proteins are usually denatured. However, under careful conditions, not only can biomolecules be kept in their native state, but complexes of multiple molecules can be kept together; by measuring the weights of these complexes, the individual components can be determined.

In the case of protein-small molecule complexes, the technique can be used to determine binding and stoichiometry (how many small molecules are bound to a given protein), and the authors discuss a number of papers in the field (including some seminal RNA-fragment examples).

Two of the authors are applying native MS to fragments at French company NovAliX. They describe screening their 350-compound fragment library against the anticancer target Hsp90 to identify 40 fragments that bind to the protein, and further characterized some of these crystallographically. The entire screen, in duplicate, required 2 milligrams of protein.

There are a few limiting issues with native mass spectrometry. First, the technique requires careful choice of buffers; in particular, detergents are not compatible. That can be a problem because omitting detergents sometimes leads to small-molecule aggregation, even with legitimate binders. The authors note that multiple binding is sometimes observed in native mass-spectrometry; it would be interesting to follow up on these observations with activity assays to determine how often these are truly non-specific or just appear so under the assay conditions.

Another issue is that the stability of protein-small molecule complexes in native mass spectrometry assays does not necessarily correlate with the (more relevant) solution-phase affinity. In the gas phase, polar interactions such as hydrogen bonds and electrostatic interactions are strengthened, while the hydrophobic effect is weakened. Intriguingly, a window into gas-phase affinity could actually be an advantage for fragment-based approaches. Polar interactions tend to be enthapically driven, while hydrophobic interactions contribute to overall affinity primarily through entropic effects. If it is true that fragments showing predominantly enthalpic binding are more attractive starting points than those whose major binding energy comes from entropy (as argued here), mass spectrometry may be a good way of finding these fragments. I don’t recall seeing a systematic study dissecting the free energies of binding of hits from native mass spectrometry into their enthalpic and entropic components. If you know of one, I would be interested to hear about it in the comments section.

1 comment:

morten_g said...

I went to biophysics meeting in Illkirch in 2006 and I seem to remember a woman, who's name I forget, mention that you got an 80-fold higher measured affinity if you had an electrostatic interaction between ligand and target. That's all I got right now.