Continuous-flow, on-line monitoring of biospecific interactions using electrospray mass spectrometry

A continuous-flow analytical screening system is presented using electrospr ay mass spectrometry to measure the interaction of biologically active comp ounds with soluble affinity proteins. The biochemical detection system is b ased on a solution-phase, homogeneous assay. In a first step, compounds to be screened (e.g., biotinylated compounds, concentration range 10-1000 nmol /L) are injected into a continuous-flow reaction system and allowed to reac t with the affinity protein (e.g., streptavidin, concentration range 3-48 n mol/L). Subsequently, a reporter ligand (fluorescein-labeled biotin 96 nmol /L) is added to saturate the remaining free binding sites of the affinity p rotein and the concentration of unbound reporter ligand is measured using e lectrospray MS in the selected-ion monitoring mode. The presence of active compounds in the sample results in an increase of the concentration of unbo und reporter ligands. The feasibility of a homogeneous MS-based biochemical assay is demonstrated using streptavidin/biotin and anti-digoidgenin/digox in as model systems. Compared to radioactive or fluorescence-based biochemi cal assays, the present assay format does not require the synthesis and pur ification of labels. Various analytical conditions were investigated to det ermine the ability of MS to measure the biochemical interactions. The avail ability of a single ligand that can be detected at 10-50 nmol/L concentrati ons by electrospray MS is sufficient to set up the biochemical assay. For t he biospecific interactions studies, detection limits of 10-100 nmol/L were obtained.