Assay Setup

The MS assay principles discussed in Sections 5.2 and 5.3 have in common that they rely on the availability of appropriate reporter molecules, i.e. chemical compounds that indicate the presence of bioactive ligands through a change of concentration. In many instances, it is not straightforward to get access to these compounds, e.g. in the case of orphan receptors where no active ligands are yet found. In this section we describe an assay format that relies on the detection of bioactive ligands after dissociation from their target protein [30]. The general principle of this reporter-free biochemical MS assay format is outlined in Fig. 5.14. The assay is based on three sequential steps, i.e. the incubation of the sample with the affinity protein, the quantitative separation of free and protein-bound compounds, and the dissociation of the protein-ligand complexes. Ligands released from the protein are subsequently detected by LC-MS.

The hardware implementation is shown in Fig. 5.15. After injecting the sample into the carrier phase, a plug of affinity protein is added during a period of 60 s, i.e. a time interval which ensures that, under the conditions described, the entire sample is able to react with the target proteins. By introducing a protein plug instead of constantly adding the target proteins to the carrier phase, a considerable reduction in affinity protein consumption is achieved. Subsequently, by implementing a short column packed with a C18 restricted-access (RA) column material (e.g. Lichrosorb ADS C18; Merck, Darmstadt, Germany), low molecular mass molecules, which did not form an affinity complex during the reaction period, are trapped inside the small hydrophobic pores of the RA beads. In contrast, the high

Fig. 5.14 Principle of label-free ligand binding MS assays. Protein (P) molecules react with the test ligand (L) to form a protein-ligand complex (PL). Unbound compounds are separated from PL by passage through a restricted-access column. Subsequently, PL is dissociated at low pH, and active ligands L are detected by LC-ESI-MS.

Fig. 5.14 Principle of label-free ligand binding MS assays. Protein (P) molecules react with the test ligand (L) to form a protein-ligand complex (PL). Unbound compounds are separated from PL by passage through a restricted-access column. Subsequently, PL is dissociated at low pH, and active ligands L are detected by LC-ESI-MS.

Lcq Deca Setup

Fig. 5.15 Analytical set-up for on-line label-free assay based on ESI-MS. MS instrument: Ion-trap mass spectrometer (LCQ Deca, Thermo Electron). P1: Carrier/HPLC pump. P2: HPLC pump delivering receptor solution. P3: HPLC pump delivering dissociation solution. P4: HPLC pump for final LC-MS analysis of released ligands. 1: Mixing union. 2: Microcoil reactor. V1: injection valve.

Fig. 5.15 Analytical set-up for on-line label-free assay based on ESI-MS. MS instrument: Ion-trap mass spectrometer (LCQ Deca, Thermo Electron). P1: Carrier/HPLC pump. P2: HPLC pump delivering receptor solution. P3: HPLC pump delivering dissociation solution. P4: HPLC pump for final LC-MS analysis of released ligands. 1: Mixing union. 2: Microcoil reactor. V1: injection valve.

molecular mass affinity proteins and complexes are allowed to pass the RA column unretained, as their size prevents diffusion into the small hydrophobic pores. Nonspecific binding of these proteins is largely avoided because of the hy-drophilic nature of the surface of the RA beads. As a result, a fast and efficient separation is achieved between those compounds that either do not or only weakly form an affinity complex with the target protein and those that do. Besides efficiently separating the bound molecules from the unbound molecules, implementation of RA columns expands the range of affinity interactions, which can be monitored using the current setup, to higher KD values as a result of the short residence times onto the RA column. As the affinity complexes typically pass the RA columns within seconds, affinity-complex dissociation becomes less pronounced when compared with other separation methods, such as size exclusion chromatography. Subsequently, after passing the first RA column, the affinity complex is subjected to a dissociation step based on a pH change, which disrupts the affinity interaction between target protein and bioactive compound. After dissociation of the affinity complex, separation of the free bioactive compound and the target protein is easily accomplished by introducing a second RA column. Li-gands, originating from the dissociated affinity complex, are trapped within the hydrophobic pores, whereas the target protein passes the RA column unretained and is directed towards waste. In this way, bioactive compounds are isolated from nonactive molecules as well as from the affinity proteins. After this loading phase, which typically takes 2 min, the second RA column is washed extensively with 1% acetic acid to remove the majority of the impurities, such as salts, originating from sources such as reagent solutions and samples. Subsequently the second RA column is switched into a 75% MeOH/2 mM ammonium acetate solution, which is constantly introduced into the ESI probe. Trapped bioactive compounds are eluted from the RA column in a well defined matrix at a flow rate of 50 mL min-1 and are subsequently analyzed by MS, using data-dependent scanning. This way, characteristic MS and MSn (n = 2 or 3) data of bioactive compounds are recorded during a single run. Molecular mass information is obtained from the MS data, whereas a mass fingerprint of the bioactive molecule is provided by MS n spectra.

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