FACS screening

FACS Screening of Combinatorial Peptide and Protein Libraries Displayed on the Surface of Escherichia coli Cells... 

After 3 1 rounds of combined MACS/FACS screening, enrichment of positive clones should be detectable. If a significant portion of the population is FACS-positive, individual clones can be labeled and analyzed as described in Protocol 7. A control should be included, in which the labeling procedure is performed without the ligand protein, to ensure that the observed interaction is ligand-specific. 

For prehminary screening and easibility studies or for rough cost estimates, one may wish to employ a version of the isothermal method which assumes that the liquid temperatures in the tower are everywhere equal to the inlet-liquid temperature. In their analysis of packed-tower designs, von Stockar and Wilke [Ind. Eng. Chem. Fun-dam. 16, 89 (1977)] showed that the isothermal method tended to underestimate the reqmred depth of packing by a factor of as much as 1.5 to 2. Thus, for rough estimates one may wish to employ the assumption that the temperature is equal to the inlet-liquid temperature and then apply a design fac tor to the result. 

The unit capacity can be determined from Fig. 19-21. Figure 19-22 can be used to determine the open-area fac tor and the slotted-opening factor F, for various screen types is given in Table 19-7. 

The ability to display proteins on the surface of an organism has been exploited as a screening method, typically in conjunction with fluorescence-activated cell sorting (FACS) [37,47,48]... 

Mass spectrometry enables the type of direct analyses described, but it does have its limitations. Online operation forces detection at infusion concentrations, in salty buffer and under complex mixture conditions. General ion suppression results from the buffer and mixture components, and mixture complexity can tax the resolution of even the best mass spectrometers. Increasing compound concentration is not the answer, as this leads to problems of solubility and increased compound consumption. We have found that the online method can work successfully for up to 100 compounds per analysis, but the false negative rate becomes appreciable [21]. As an alternative for ligand discovery purposes, we have developed a FAC-LC/MS system in which FAC effluent is sampled and analyzed by LC/MS [19]. This system offers the ability to concentrate mixture components and introduces another dimension to the data in order to tolerate more complex mixtures (Fig. 6.9). Using this system, we have screened approximately 1000 modified trisaccharide acceptor analogs targeting immobilized N-... 

Fig. 6.9 Schematic of FAC effluent sampling strategy for insertion of an LC/MS step to increase rugged ness of the discovery mode of analysis, as applied to high throughput screening for ligands to GnT-V [19]. The insets represent LC/MS data for a strong ligand for four fractions of FAC effluent (1, x,...

Table 6.2 Summary of the hit data obtained from the CnT-V screening using the FAC-LC/MS method [19], Hits are categorized based on an arbitrary binning strategy, where breakthrough timesa are converted into approximate (fj values. Adapted with permission from the American Chemical Society.

Fig. 6.11 Using rollups to efficiently prescreen mixtures for the presence of "hits". In this example, six mixtures of approximately 90 compounds each (A-E) were screened in a dual protein FAC assay (/S-galactosidase, GS1B4). The dashed red and blue curves in each chromatogram represent the breakthroughs of the /S-galactosidase and GSl B4 indicators, respectively, in the absence of the...

From this discussion, there is an obvious advantage to FAC in that the assay development approach is extremely flexible and adaptable to the requirements of the interaction to be studied. It is worth mentioning that the effort placed on creating a FAC cartridge is never wasted - it can be used as a simple capture/elute tool for alternative screening approaches, and even in preclinical studies that require methods for monitoring drug candidates and their active metabolites [30]. 

More recently, Brennan has shown that FAC-MALDI-MS can be used to screen small molecules, relying upon MRM transitions to overcome the chemical noise generated by the matrix [16]. This is an acceptable approach for known compounds, but for ligand discovery from uncharacterized mixtures, ion selection... 

As indicated above, one major advantage of bacterial and other cell-based surface display formats lies in the ability to use fluorescence-activated cell sorting for high-throughput library screening. With modem FACS equipment, such as the Cytomation MoFlo or the FACS Vantage from Beckton-Dickinson, sorting rates of up to 100 000 events per second are possible [10]. 

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