Affinity selection-mass spectrometry[edit]

While adoption of affinity selection-mass spectrometry (AS-MS) has led to an expansion

of assay formats,[18] the general technique follows a simple scheme. Protein targets are incubated
with small molecules to allow for the formation of stable ligand-protein complexes, unbound small
molecules are removed from the mixture, and the components of remaining ligand-
protein complexes are analyzed using mass spectrometry.[18] The bound ligands identified are
then categorized as hits and can be used to provide a starting point for lead generation. Since
AS-MS measures binding in an unbiased manner, a hit does not need to be tied to a functional
readout, opening the possibility of identifying drugs that act beyond active sites, such
as allosteric modulators and chemical chaperones, all in a single assay.[19] Because small
molecules can be directly identified by their exact mass, no derivatization is needed to confirm
the validity of a hit.[19] Among derivatization- and label-free approaches, AS-MS has the unique
advantage of being amenable to the assessment of multiple test compounds per experiment—as
many as 20,000 compounds per experiment have been reported in the literature, and one group
has reported assaying chemical libraries against heterogeneous protein pools.[19] The basic steps
of AS-MS are described in more detail below.

Affinity selection via gel filtration. A pool of test compounds is added to a protein sample, which is passed through a gel

filtration column. Protein-bound compounds move around the beads and exit the column quickly. Unbound compounds are

small enough to travel through beads and take a longer path before elution. This image was made using BioRender.com.

Affinity selection[edit]
A generalized AS-MS workflow begins with the pre-incubation of purified
protein solutions (i.e. target proteins) with chemical libraries in microplates. Assays can be
designed to contain sufficiently high protein concentrations to prevent competition for binding
sites between structural analogs, ensuring that hits across a range of affinities can be identified;
inversely, assays can contain low protein concentrations to allow for distinction between high and
low affinity analogs and to inform structure-activity relationships.[19] The choice of a chemical
library is less stringent than other high-throughput screening strategies owing to the lack of
functional readouts, which would otherwise require deconvolution of the source compound that
generates biological activity. Thus, the typical range for AS-MS is 400-3,000 compounds per
pool.[19] Other considerations for screening are more practical, such as a need to balance
desired compound concentration, which is usually in the micromolar range, with the fact that
compound stock solutions are typically stored as 10 millimolar solutions, effectively capping the
number of compounds screened in the thousands.[19] After appropriate
test compounds and targets are selected and incubated, ligand-protein complexes can be
separated by a variety of means.[18]
Separation of unbound small molecules and ligand-protein complexes[edit]
Affinity selection is followed by the removal of unbound small molecules via ultrafiltration or size-
exclusion chromatography, making only protein-bound ligands available for downstream
analysis.[18] Several types of ultrafiltration have been reported with varying degrees of throughput,
including pressure-based, centrifugal, and precipitation-based ultrafiltration.[18] Under both
pressure-based and centrifugal formats, unbound small molecules are forced through
a semipermeable membrane that excludes proteins on the basis of size. Multiple washing steps
are required after ultrafiltration to ensure complete removal of unbound small molecules.
[18]
 Ultrafiltration can also be confounded by non-specific adsorption of unbound small
molecules to the membrane.[18] A group at the University of Illinois published a screening strategy
involving amyloid-beta, in which ligands were used to stabilize the protein and prevent its
aggregation. Ultrafiltration was used to precipitate aggregated amyloid-beta and remove
unbound ligands, while the ligand-stabilized protein was detected and quantified using mass
spectrometry.[18]
Size-exclusion chromatography (SEC) is more widely used in industrial drug discovery and has
the advantage of more efficient removal of unbound compounds as compared to ultrafiltration.
[18]
 Size-exclusion approaches have been described in both high-performance liquid
chromatography (HPLC) based and spin column formats.[18] In either case, a mixture of
unbound ligands, proteins, ligand-protein complexes is passed through a column of porous
beads. Ligand-protein complexes are excluded from entering the beads and exit
the column quickly, while unbound ligands must travel through the beads and are retained by
the column for a longer time. Ligands that elute from the column early on are therefore inferred to
be bound to a protein.[18] The automated ligand identification system (ALIS), developed
by Schering-Plough, uses a combined HPLC-based SEC to liquid chromatography-mass
spectrometry (LC-MS) system that separates ligand-protein complexes from
unbound ligands using SEC and diverts the complex toward an LC-MS system for on-line
analysis of bound ligands.[18] Novartis' SpeedScreen uses SEC in 96-well spin column format,
also known as gel filtration chromatography, which allows for simultaneous removal of
unbound ligands from up to 96 samples.[20] Samples are also passed through porous beads,
but centrifugation is used to move the sample through the column.[20] SpeedScreen is not coupled
to an LC-MS system and requires further processing prior to final analysis. In this
case, ligands must be freed from their targets and analyzed separately.
Analysis of bound ligands[edit]
The final step requires bioanalytical separation of bound ligands from their targets, and
subsequent identification of ligands using liquid chromatography-mass spectrometry.[19] AS-MS
offers means for identifying small molecule-protein interactions either directly - through top-down
proteomic detection of intact complexes - or indirectly - through denaturation of small molecule-
protein complexes followed by identification of small molecules using mass spectrometry.
[18]
 The top-down approach requires direct infusion of the complex into an electrospray
ionization mass spectrometry source under conditions gentle enough to preserve the interaction
and maintain its integrity in the transition from liquid to gas.[18] While this was shown to be
possible by Ganem and Henion in 1991, it is not suitable for high throughput.
[18]
 Interestingly, electron capture dissociation, which is typically used in structure elucidation
of peptides, has been used to identify ligand binding sites during top-down analysis.[18] A simpler
method for analysis of bound ligands uses a protein precipitation
extraction to denature proteins and release ligands into the precipitation solution, which can then
be diluted and identified on an LC-MS system.
An example target identification by chromatographic co-elution (TICC) workflow.  Drug-

spiked lysate is fractionated using ion exchange chromatography. Fractions are collected every minute, then analyzed for both

drug and protein content using LC-MS/MS. Drug and protein elution profiles are constructed and correlated. Target

identification is supported by a strong correlation in elution profile between a drug and a protein.  This image was made

using BioRender.com.

Target identification by chromatographic co-elution (TICC)[edit]

Target identification by chromatographic co-elution does not rely on differences in protein stability
after drug treatment. Instead, it rests on the assumption that drugs form stable complexes with
their target proteins, and that those complexes are robust enough to survive a chromatographic
separation. In a typical workflow, a cell lysate is incubated with a drug, then the lysate is injected
onto an ion-exchange chromatography system and fractionated. Lysate proteins are eluted along
an ionic strength gradient and fractions are collected over short time intervals. Each fraction is
analyzed by LC-MS/MS for both protein and drug content. Individual proteins elute with
characteristic profiles, and pre-incubated drugs should mirror the elution profiles of the targets
they complex with. Correlation of drug and protein elution profiles allows for targets to be
narrowed down and inferred.[21]