Native state electrospray ionization mass spectrometry (ESI-MS) is, in theory, a fast and easy way to find fragments: just mix protein with fragment, shoot it on the MS, and look for complex. As a bonus, the exact mass tells you whether your fragment is what you think it is (or at least whether it has the right mass). However, published examples are relatively rare, and not always favorable. A new paper in J. Med. Chem. by Tom Peat, Sally-Ann Poulsen, and their colleagues at CSIRO and Griffith University seeks to change this.
The researchers chose the fragment-friendly model protein carbonic anhydrase II (CA II) as their target. They first screened a library of 720 fragments, each at 100 µM, using surface plasmon resonance (SPR). This yielded 7 hits, with affinities ranging from 1.35 to 1280 µM. These seven hits were then assessed by ESI-MS using equimolar concentrations of protein and fragment (10 or 25 µM each). Encouragingly, all seven hits confirmed. Soaking these fragments into crystals of CA II yielded structures for six of them.
This is nice, but of course the real question is how well ESI-MS works as a primary screen. To address this, the researchers chose 70 compounds structurally related to the 7 hits and independently tested these using both SPR and ESI-MS. This yielded 37 hits, of which 24 were detected both by SPR and ESI-MS. In fact, every SPR hit was confirmed by ESI-MS. Of 14 fragments subsequently soaked into crystals of CA II, 7 provided interpretable electron density.
This is impressive, and the researchers note that the level of agreement between SPR and ESI-MS might be better still, since some of the ESI-MS hits did give signals by SPR – they were just weaker than the chosen cutoff (KD ≤ 3 mM). Thus, in contrast to a paper discussed last year, ESI-MS does seem to be a sensitive detection method. In fact, given the low concentration of fragment needed, the researchers suggest that it could be useful for screening fragments with lower solubilities.