High Resolution Screening (HRS) is a hyphenated screening technique which combines High Performance Liquid Chromatography (HPLC) with an on-line post-column bioassay and parallel mass spectrometry detection. HRS is an excellent analytical technique for rapidly pinpointing bioactive peptides in complex mixtures such as animal venoms and other natural extracts.
There have been various types of biochemical assays successfully applied in HRS systems, however functional cell-based bioassays were out of scope until now. This work describes the development of a cell-based HRS methodology using flow cytometry for a direct post column cellular bioassay readout. With a post column split, next to the on-line flow cytometry readout, also mass spectrometry data is obtained in parallel for measurement of the accurate masses of the bioactive peptides eluting.
The α7-nicotinic Acetylcholine Receptor (α7-nAChR) is a potential therapeutic target for several central nervous system (CNS) related diseases. A functional calcium-flux assay with human neuroblastoma SH-SY5Y cells over-expressing the α7-nAChR was used to screen for bioactive peptides in venoms and other natural extracts. The analytical system was first optimized and pharmacologically validated in agonist, allosteric modulator and antagonist setup. Next, various types of natural extracts were screened, such as a tobacco extract in agonist mode as a proof of principle, and then snake venoms in mixed antagonist and agonist analysis mode. Eluting bioactive peptides could directly be correlated to their respective accurate mass. The developed methodology opens up the possibility for direct and fast post column cellular screening of venoms for bioactive peptides targeting the α7-nAChR. It is anticipated that other types of fast cell-based assays (e.g. ion flux assays in general) are also applicable to the here presented analytical technique.