08 June 2015

Benchmarking native mass spectrometry

Mass spectrometry (MS) is one of the less common tools to find fragments. In the conceptually simplest approach (native mass spectrometry), you incubate your protein with a putative ligand and ionize the mixture. Fragment binding is detected by an increased mass for the complex, and the strength of binding by the ratio of heavier bound complex peak to protein peak. However, the liquid to gas phase transition is a big step, and often the complex does not survive. Aside from more specialized applications of MS (such as herehere, and here) there aren’t many published examples. A recent paper from Federico Sirtori and colleagues at Nerviano and Università degli Studi di Milano in Eur. J. Pharm. Sci. describes fragment screening by native MS in detail.

The researchers used the reliable model protein Hsp90, which was also used in a previous MS study and in benchmarking other techniques. One of the many benefits of Hsp90 is a wealth of well-characterized inhibitors with a range of affinities, and these were used to calibrate the technique. This turned out to be critical: beyond sample preparation itself (beware non-volatile buffer components), all kinds of parameters can be adjusted including various voltages, temperatures, vacuum strength, and ion source. Get one of these wrong and your non-covalent complex either fails to ionize or blows apart.

In addition to using published data on known compounds, the researchers ran both fluorescence polarization (FP) and surface plasmon resonance (SPR) assays to independently determine dissociation constants. Initially the results from MS (a Q-TOF) were quite different, but after optimization the team was ultimately able to find conditions that gave qualitatively as well as quantitatively similar results for ligands with affinities ranging from picomolar to ~100 micromolar.

Thus encouraged, the team embarked on a fragment screening campaign. The Nerviano fragment library consists of 1914 molecules mostly following the rule of 3, though halogenated fragments up to 380 Da are allowed as are compounds with up to 6 hydrogen bond acceptors. The fragments were run in mixtures of 5, with protein at 2.5 µM and each compound at the low concentration of 10 µM. Sample injection and data processing were automated, and the entire screen took 2 days and 2 mg of protein.

Given the low concentration of fragments, the researchers lowered the bar for potential hits, yielding 282 compounds. These were retested individually, yielding 146 confirmed hits that gave signals of 5.2-29.7% bound protein. This is a high hit-rate, particularly given that these binding levels suggest affinities in the 20-179 µM range. Indeed, only 5 fragments could be competed by a high-affinity binder, suggesting either that the others bind outside the active site or are non-specific (false positives). Regarding false negatives, Nerviano reported the results of an NMR fragment screen against Hsp90 last year, and 12 of 14 hits identified there could also be detected by MS. The other two were likely below the detection limit of the MS assay.

Unfortunately, the researchers do not discuss thermodynamics. In theory enthalpic interactions dominate over entropic interactions in the gas phase, but it is unclear whether any of the observed binders were strongly entropy-driven.

In the end, it appears that fragment screening by native MS is workable, but the sensitivity is probably lower than other techniques. Of course, increasing the ligand concentration would increase the sensitivity to weaker binders, but at the cost of more non-specific binding – which is already considerable. Also, Hsp90 is about the friendliest protein one can imagine. I would be reluctant to try this with a more challenging target that lacks good tool ligands. But if you want to give it a go, this paper provides a wealth of information for getting started. And if you have experience with native MS, please share it in the comments.


Dr. Teddy Z said...

I really really like native MS. I have seen it work where SPR outright fails. There are some tricks to the trade of course. It has some significant advantages over the other direct detect biophysical methods: protein QC every experiment and the ability to do stoichiometry for example.

Valerie Vivat said...

Non-specific adduction occurring during electrospray has been largely documented by Klassen's group (see a nice review from Kitova et al. published in JASMS (2012) "Reliable determination of Protein-Ligand interactions by direct ESI-MS measurements. Are we there yet?"). Artefactual adduction of compound to protein during solvent evaporation, concentration and fission of the charged dropplets typically show Poisson-like distribution. This phenomenum is prone to occur with large excess of compound in incubation mixtures.
Therefore screening for low affinity hits, in the three digit µM range for example (typically fragment molecules), using native MS, absolutely requires high affinity tool molecule to be available to distinguish true hits from false positives. From our experience a screen of 1500 fragment molecules (MW ~ 200) against a nuclear hormone receptor target produced about 3.5% validated hits. Binding stoichiometry and reversibility as well as an estimation of the binding affinity range were also directly available from mass spectra. Screen was done side by side against the apo receptor and against the receptor saturated with the tool compound.
Regarding false negative due to in source dissociation of the complex, the group of John Klassen has described additives and indirect approach that may be beneficial (see above review).

Dan Erlanson said...

Thanks Teddy and Valerie. I do like the idea of native MS, I just don't recall seeing many examples where it excels in true discovery mode as opposed to proof of concept.

It's true that native MS does give you protein QC, though this can be a limitation if you're working with, for example, glycosylated protein.

Also, you can get stoichiometry with SPR, though it requires more effort. That said, I wonder how often apparent stoichiometry observed with native MS is actually just non-specific binding.

I did notice that while the NovAliX biophysics conference going on right now has two scheduled presentations on HDX MS, there do not appear to be any on native MS (though I don't have access to the poster titles).