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ALIS System Design

All the chromatography steps of the ALIS process are accomplished using a single suite of hardware. As described in reference [38], in the ALIS system the SEC and RPC-MS systems are directly coupled through a single valve. This strategy reduces sample loss due to non-specific interaction with multiple surfaces or through additional sample transfer steps, allowing maximum sample recovery, improved system reliability, and good sample-to-sample reproducibility. Also, since sample handling and tracking are minimized, improved workflow efficiency enables a highly automated, ''screening sample-to-results'' process.

ALIS uses continuous SEC to isolate protein-ligand complexes from unbound library members. Samples containing a target protein, protein-ligand complexes, and unbound compounds are injected onto an SEC column, where the complexes are separated from non-binding components by a rapid SEC step. As shown in Fig. 3.2, the SEC column eluate is monitored using UV detectors to confirm that the early-eluting protein fraction, which elutes in the void volume of the SEC column, is well resolved from unbound components that are retained on the column. After the peak containing the protein and protein-ligand complexes elutes from the primary UV detector, it enters a sample loop where it is excised from the flow stream of the SEC stage and transferred directly to the LC-MS via a valving mechanism. A second UV detector positioned after the valve records the SEC components not delivered to the LC-MS. The primary detector shows the separation of the protein peak from unbound library members, and by comparing the

Fig. 3.2 SEC chromatograms from typical ALIS experiments. (A) UV responses from detectors positioned before and after a sampling valve show that the protein-ligand complex, eluting at 20 s, is excised from the SEC stream for transfer to RPC-MS. (B) An overlay of ten SEC chromatograms demonstrates ALIS sample-to-sample reproducibility. Reprinted from [40] with permission from Elsevier.

Fig. 3.2 SEC chromatograms from typical ALIS experiments. (A) UV responses from detectors positioned before and after a sampling valve show that the protein-ligand complex, eluting at 20 s, is excised from the SEC stream for transfer to RPC-MS. (B) An overlay of ten SEC chromatograms demonstrates ALIS sample-to-sample reproducibility. Reprinted from [40] with permission from Elsevier.

two UV detector outputs, an operator can determine whether any unbound library members might have been introduced to the MS. Another important operational advantage of this configuration is demonstrated in Fig. 3.2B. In this screenshot, an overlay of SEC chromatograms from a series of ALIS experiments shows good symmetry and reproducibility for the protein peak, indicating no deleterious interactions with the library or sample preparation that may cause misshapen or absent protein peaks.

Following the SEC stage, the band containing protein-ligand complexes is immediately transferred to an RPC column where ligands are dissociated from the protein and trapped on the RPC stationary phase. The dissociated ligands are eluted into a mass spectrometer for analysis, and automated software algorithms search the mass spectral data to identify the ligands by their molecular weight. ALIS reports only compounds that bind directly to the target of interest, preventing false positives that arise from off-target activity or interactions with substrates or other reagents. Since ALIS directly identifies bound components by MS, the incidence of false positives based on ''bulk effects'' and non-specific binding is lower than that of biochemical assays that yield a secondary readout of activity.

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