18 March 2015

Mass Spec Screening in Solution

Mass spectrometry is a technique that most people are familiar with, as a QC tool.  It also has been demonstrated as a screening/validation tool.  Native mass spectrometry (nMS) has been discussed here, Weak Affinity Chromatography (WAC) here, and Hydrogen-deuterium exchange (HDX) here.  All of these methods have advantages and disadvantages.  A "new" method is the ligand-observed MS screening (LO-MS).  [I put new in quotes because I know of at least one company that has been using this method for screening for years via a CRO.]

The concept of LO-MS is straight forward (Figure 1) and very similar to WAC.  A mixture of fragments, in this case 384, are mixed with target (NS5B), incubated, and the ultrafiltrated (50kDa cutoff).  This step eliminates the need for the immobilization step in WAC, ensuring the native conformation.  The fragments were at 25 uM, while the target was at 50 uM. 
Figure 1.  Fragments MW 165 and 130 are binders.  MW162 and 150 are not. 
Retained fragments are then dissociated with 90% methanol and those showing intensity higher than the protein-minus control are considered binders (S/N  greater than 10).  In their library, 5% of the compounds were not amenable to mass spec detection, but they included them to increase the complexity of the mixture.  In the end, they ended up with 20 binders in 20 minutes!  They repeated the screen with smaller mixtures (50 and 84 fragments) where they found 12 binders (a subset of the original 20).  As a follow up, they ran the binders by SPR, validating 10 of the binders (50%).  5 out of these 10 gave useable crystals (observable electron density for the fragment) (50%).  They also show how the data can be used to generate Kds (like WAC).

This method raises some issues with me, but first let me say, it sure seems to work, and fast to boot.  From people I know who have used this to screen, they have been very happy.  Here is what bothers me: self-competition in the tube a discussed here and here, this is a non-equilibrium method (variable protein concentration during the ultrafiltration), and it is an indirect method.  For me, I prefer methods that directly detect ligand-target interactions, like NMR, SPR, and nMS.


Anonymous said...

The SPR sensograms in the supplemental are 'interesting'

Dr. Teddy Z said...

That's being generous. http://www.nature.com/srep/2015/150210/srep08361/extref/srep08361-s1.pdf

Anonymous said...

David Myszka would be turning in his grave, if he were dead that is.

Another problem is that I imagine it would be a poor method to determine binders with 1:1 stoichiometry, and thus would preferentially pull out non specific binders. Perhaps the SPR could be used as evidence of this.