Pulsed Ultrafiltration MS

Pulsed ultrafiltration MS (PUF-MS) represents an inline high throughput affinity screening method with a variety of potential uses in the discovery and development of pharmaceuticals [22]. The in-line combination of solution-phase equilibration, ultrafiltration, and electrospray liquid chromatography mass spectrometry (LC-ESI-MS) facilitates the identification of high affinity target-specific

Ultrafiltration Mass Spectrometry

Fig. 4.4 Scheme of pulsed ultrafiltration— mass spectrometry (PUF-MS) to screen chemical mixtures for compounds that bind to a macromolecular receptor. The ultrafiltration membrane traps a receptor in solution, but allows low molecular weight compounds to pass through. Bound ligands are eluted from the chamber by destabilizing the ligand-receptor complex with an organic solvent or pH change. The ligands are characterized with MS. Included from [22] with permission from Wiley Periodicals.

Fig. 4.4 Scheme of pulsed ultrafiltration— mass spectrometry (PUF-MS) to screen chemical mixtures for compounds that bind to a macromolecular receptor. The ultrafiltration membrane traps a receptor in solution, but allows low molecular weight compounds to pass through. Bound ligands are eluted from the chamber by destabilizing the ligand-receptor complex with an organic solvent or pH change. The ligands are characterized with MS. Included from [22] with permission from Wiley Periodicals.

ligand(s), and also allows for potential reuse and/or recovery of the target proteins. PUF-MS has been used for rapid screening of several drugs to determine their effect on metabolism, and to characterize various primary metabolites (i.e., microsomal cytochromes P450 [21]). During PUF-MS, soluble target is equilibrated with modest compound mixtures of @20 molecules for approximately 20 min and injected into the ultrafiltration chamber (the ''pulse''). As shown in Fig. 4.4 [21], the target protein is trapped in solution on one side of the chamber by an ultrafiltration porous membrane of defined mass selectivity (i.e., a 10 kDa molecular weight cutoff). The sample is then flushed for a predetermined amount of time (8-10 min) with water to remove unbound ligands. Van Breemen and colleagues demonstrated that during the washing step more than 98% of the unbound compounds diffused out from the ultrafiltration chamber, reducing their concentration to background levels in the electrospray mass spectra. The wash may be discarded to waste or monitored continuously by the mass spectrometer (Fig. 4.4). Next, the ligand-target complex in the mobile phase is disrupted by addition of organic solvent (i.e., 50:50 v/v methanol:water) or pH changes, thereby releasing bound ligand(s) into the mass spectrometer for identification. In this manner, the ultrafiltration chamber functions as a solution-phase extraction device.

In a continuous infusion mode, the mass spectrometer acts as the detector for target-specific small molecules exiting the sample chamber. Each compound's intrinsic unique mass results in specific elution profile that is recorded for quanti-

tation. Specifically, by integrating the area under each spectra curve for a given compound's mass signal the total amount of target-specific ligand can be calculated. If the starting concentrations of a ligand and target protein are known, then one can calculate the compound's relative KD for that target. Hence, both kinetic and thermodynamic parameters can be simultaneously deduced from these ligand-binding studies for multiple ligands against a single protein target.

Similar to ASMS, in pulsed ultrafiltration screening assays it is important to keep the target concentration in excess of the compound concentration. In general, a protein concentration is chosen to be approximately equal to the KD of the weakest ligands. For example, the use of 1 mM protein permits the detection of target-specific binders that exhibit KD values of 0.1-1.0 mM. The ratio of receptor and ligand concentrations, or selection stringency, determines the number of "hits" that might be obtained when screening large compound mixtures. High receptor concentrations typically result in larger numbers of hits because weaker ligands will be identified together with the high-affinity compounds. With screening libraries that contain large mixtures, even with diverse structures, excess protein is required to minimize competition between ligands so that all of the potential hits may be detected. When this experiment is conducted in the presence of a high molecular weight protein, elution of a compound with no target-specific affinity (non-binder) follows the same profile as without target. However, if affinity exists between a compound and the target, its elution profile is perturbed. A caveat, though, is that if the binding reaction exhibits a very rapid off rate the total area under the curve for that compound is unaltered. Binding-induced shifts in a compound's elution profile over time are interpreted in terms of the binding affinity [47].

There are several advantages and disadvantages with PUF-MS with respect to other affinity selection techniques. In contrast to ASMS, the equilibration and filtration steps are coupled to the mass spectrometer, and the rate limiting steps therefore are the lengthy equilibration time required for ligand binding and wash time to remove unbound ligands. For precious protein samples, however, an advantage is that protein usage can by minimized in this inline technique. If the protein can be treated in a manner that releases bound ligands but does not irreversibly denature the protein (for example, by careful choice of organic solvent or pH shift), then the protein may be used repeatedly. Additionally, compound handling steps are minimized in an inline procedure and the potential for compound loss to surfaces and introduction of adventitious contaminants is minimized. In addition, like ASMS, affinity selection reactions occur in solution; screening covalently immobilized proteins or ligands can compromise protein and/or ligand native conformations or binding characteristics. A disadvantage that PUF-MS shares with ASMS is the proclivity for non-specific binding of small molecules to the ultrafiltration membrane. This necessitates certain controls. Specifically, because the elution of a given binder from the chamber is slowed by reversible protein association and dissociation, relative to controls performed in the absence of protein, the elution profiles of such a binder differ between these two cases. Using differential equations that describe solution fluidity and ligand-

target association, quantitative thermodynamic and kinetic information can be derived from the degree of difference between their elution profiles [47, 48].

Finally, several features inherent to PUF-MS suggest this methodology is a potentially powerful tool in new drug discovery. Predominantly, it has been demonstrated to be applicable to ''reverse pharmacology'' studies in which a given receptor of interest has been identified and isolated, but novel small molecules that bind to the receptor are needed [22]. Also, the binding behavior of these ligands can be quantitatively measured (association constants or binding rates). There is also evidence that PUF-MS is very effective for metabolic screening [49]. van Breemen and colleagues accurately identified novel phase I metabolites of xeno-biotic compounds generated in the presence of cytochromes P450. Also, pulsed ultrafiltration was used to screen four natural products extracts for the metabolic formation of electrophilic quinoid metabolites [22]. Using tandem MS approaches, the chemical diversity of the mixtures did not compromise the ability of PUF-MS to detect such reactive metabolites because tandem MS can selectively detect fragment ions from glutathione adducts, using neutral-loss scanning or precursor-ion scanning [22]. These applications demonstrate the versatility of PUF-MS and are likely to be valuable in new drug discovery endeavors.

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