Pulsed ultrafiltration

Figure 1.4 High-field (aliphatic) regions of 500 MHz single-pulse H-NMR spectra of an inflammatory synovial fluid ultrafiltrate sample obtained before (a) and after equilibration (b) with 2.00 x 10 mol/dm H2O2 at ambient temperature for a period of 24 h. Typical spectra are shown. For abbreviations, see Fig. 1.2 with acac,...

Zhao, Y.-Z., van Breemen, R.B., Nikolic, D., Huang, C.-R., Woodbury, C.P., Schilling, A., Venton, D.L. Screening solution-phase combinatorial libraries using pulsed ultrafiltration/electrospray mass spectrometry. J. Med. Chem. 1997, 40, 4006-4012. 

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... 

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... 

Johnson BM, Nikolic D, van Breemen RB Applications of pulsed ultrafiltration-mass spectrometry. Mass Spectrom Rev 2002, 21, 76-86. 

Schilling A, Venton DL Screening solution-phase combinatorial libraries using pulsed ultrafiltration/ electrospray mass spectrometry. J Med Chem 1997, 40, 4006-4012. 

Screening for inhibitors of dihydrofolate reductase using pulsed ultrafiltration mass spectrometry. Comb Chem High Throughput Screen 1998, 1, 47-55. 

Utility of pulsed ultrafiltration-mass spectrometry, where microsomal fractions are entrapped in stirred ultrafiltration chambers and the output of the chamber is introduced directly into an electrospray mass spectrometer shows particular promise [37]. 

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],... 

Venton DL, Pulsed ultrafiltration mass spectrometry a new method for screening combinatorial libraries, Anal. Chem., 69 2159-2164, 1997. 

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... 

Figure 7.12 Off-bead structure determination pulsed ultrafiltration/ESI mass spectrometry.

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. 

The second solution to increasing the throughput of pulsed ultrafiltration mass spectrometry has been to miniaturize the ultrafiltration chamber volume while maintaining the flow rate and chamber pressure. Because the ultrafiltration membrane cannot withstand high pressure without rupturing, the ultrafiltration process cannot be accelerated simply by increasing the flow rate through the chamber. The approach of Beverly et al. (72) was to fabricate a 35-/u,L ultra-... 

The advantages of pulsed ultrafiltration-mass spectrometry include the variety of different applications that may be carried out, the convenience of on-line screening, solution-phase screening, the ability to screen either combinatorial libraries or natural product extracts, the diversity of receptors that may be screened, and the freedom to use either volatile or non-volatile binding buffers. For metabolic and toxicity screening, flow injection analyses have the additional advantages that product feedback inhibition is prevented so that the metabolic profile more closely approximates the in vivo system ( 70). Finally, the... 

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