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 

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- 

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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... 

Other purified enzyme assays include quantitative measurements of oxygen consumption with a Clark electrode in prostaglandin biosynthesis [108], radioimmunoassay (RIA) [109], enzyme immunoassay (EIA). [110,111], and a pulsed ultrafiltration liquid chromatography-mass spectrometry (LC-MS) binding assay [112],... 

As a case example we will consider the approach of van Breemen and co-workers (67-69), who applied pulsed ultrafiltration ESI-MS (70) to the screening for inhibitors of adenosine deaminase (67, 68) and dehydrofolate reductase (69). A schematic representation of the whole process is repiorted in Fig. 7.12. The authors used an HPLC/MS apparatus where the column was substituted with an ultrafiltration chamber (around 100 pL volume) equipped with a 10,000-MW cutoff membrane. Aqueous buffer solutions of the library components and the receptor were incubated, then loaded... 

Pulsed Ultrafiltration-Mass Spectrometry. A versatile approach to screening solution phase combinatorial libraries and natural product extracts is pulsed ultrafiltration-mass spectrometry (61,62), which uses a standard LC-MS system with an ultrafiltration chamber substituted for the HPLC column. 

Combinatorial chemistry initiatives have created a tremendous challenge for activities that deal with the screening of these mixtures for activity against a specified target. MS-based approaches that use affinity selection, encoding methodologies, pulsed ultrafiltration, and anti-aggregatory approaches have been described. 

Figure 9.2 Identification of er7f/rra-9-(2-hydroxy-3-nonyl)adenine as the highest affinity ligand for adenosine deaminase in a combinatorial library of 20 compoimds using pulsed ultrafiltration electrospray MS. Reprinted from [39] with permission. 1997, Amerioan Chemieal Society.

FIGURE 2.7 Scheme illustrating the pulsed ultrafiltration ESI-MS assay for screening COX-2 inhibitors. (Reprinted from Nikolic et al. [58], used with permission. Copyright 2000 by the American Chemical Society.)... 

Pulsed Ultrafiltration-Mass Spectrometry in this approach, the receptor was placed in an ultrafiltration membrane, and a pulse of library compounds was applied to this cell [104], The active components were retained in the ultrafiltration cell, and the unretained components and the binding buffer were washed away to the waste. The bound components were liberated and eluted from the membrane with a flow of methanol or an acidic solvent. ESI-MS or ESI-MS/MS provided the identity of the components liberated. 

The development of on-line combination of pulsed ultraliltration (PUF) and MS was reported by Van Breemen et al. [79-80]. Relevant CYP substrates, such as imipramine and chlorpromazine, were flow-injected into an ultrafiltration chamber, where rat liver microsomes were trapped in. Negative-ion ESl-MS was applied for the on-line detection of the metabolites. 

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