Native mass spectrometry (
nMS) is
one of the less commonly used fragment-finding
methods. The approach entails
mixing proteins and ligands and gently ionizing them under non-denaturing
conditions to look for complexes. As with many other methods, multiple fragments
can be screened in a single sample. In a new
ACS Med. Chem. paper, Ray
Norton and collaborators at Monash University and CSIRO report screening multiple
proteins in a single sample.
The researchers were interested
in fatty acid-binding proteins, or FABPs. As their name suggests, these
transporter proteins shuttle lipophilic molecules such as fatty acids around
cells. The ten human isoforms are expressed in different tissues and have
different functions in metabolic signaling, but their similarity to one another
has made finding selective
chemical probes difficult. Enter nMS.
FABP isoforms 1-5 are the most
heavily studied, and these were first assessed individually. They ionized well, though in some cases peaks corresponding to both the native
protein and a complex with acetic acid was observed, not surprising given that the
buffer contained 50 mM ammonium acetate.
Next, all five proteins were
mixed together at 10 µM each. All the proteins could still be observed (with or
without bound acetate), though some proteins did give stronger signals than
others due to differences in ionization efficiency.
Adding small molecule WY14643,
which the researchers had previously found to bind to FABPs in a fluorescence
polarization (FP) assay, led to a more complex spectrum, with peaks corresponding
to unbound proteins, proteins bound to WY14643, proteins bound to acetate, and
proteins bound to both acetate and WY14643. When WY14643 was added at 10 µM,
the selectivity profile was consistent with the FP data. Interestingly though,
when ligand was added at the total concentration of all protein isoforms (50 µM),
the selectivity profile changed. The researchers suggest this may be due to nonspecific
binding at higher ligand concentrations, as has been seen
previously for nMS.
To explore the generalizability of
multiplexing nMS, the researchers turned to more potent (nanomolar) ligands. As
with WY14643, these molecules showed good agreement with published selectivity rankings
at lower ligand concentrations with some non-specific binding at higher
concentrations.
When I first
wrote about nMS back
in 2010, I noted that “the stability of protein-small molecule complexes in
native mass spectrometry assays does not necessarily correlate with the (more
relevant) solution-phase affinity,” and this fact is investigated in the paper.
Careful optimization of the experimental conditions, including ionization voltage
and temperature, led to good relative selectivity rankings for a given ligand
across the different FABP isoforms but differences in absolute values from
those measured by
ITC.
Another challenge is the fact
that the five FABP isoforms tested have similar molecular weights; in one case
a ligand complexed with FABP3 was difficult to distinguish from free FABP2. The
researchers could solve this by using different protein constructs, such as a hexa-histidine-tagged
version of FABP3.
Overall this is an interesting
approach, and the paper does an excellent job describing the technical details
and limitations. Along with
protein-observed 19F NMR, mass spectrometry is a rare experimental technique suitable for screening mixtures of
proteins in solution. Indeed, this becomes even easier when screening covalent binders,
as seen in this
paper from 2003, since there is no need to worry about ligand
dissociation during ionization. And with the increasing interest in covalent drugs, the use of MS is only likely to increase.
