Direct Dynamic Protein-Affinity Selection Mass-Spectrometry.

Chromatographia
Niels JonkerHubertus Irth

Abstract

A new methodology is described enabling the affinity screening of potential ligands towards the human estrogen receptor alpha ligand binding domain (ERalpha-LBD). In-solution incubation is performed of the analyte and the His-tagged ERalpha-LBD. The bound complex is immobilized on a nickel-loaded protein-affinity selection column, where after the unbound fraction is removed. The immobilized protein-ligand complex is exposed to a decreased pH value and an increased organic modifier concentration releasing the ligand for MS detection, and precipitating the proteins on a filter positioned between the affinity column and the mass spectrometer. The trapping column can be regenerated for reuse at least 70 times. The advantages of the methodology over existing methodologies are the absence of a pre-concentration as well as a chromatographic separation step, resulting in a significantly shorter analysis time compared to previously described procedures, and in addition, allowing the determination of solutes with unfavorable chromatographic properties. The overall analysis time now can be reduced about 250% to approximately 6 min. Replacing the filters after every measurement results in an intra-day standard deviation of 14.8% and an int...Continue Reading

References

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May 23, 2009·Chemistry : a European Journal·Niels EldersRomano V A Orru
Mar 1, 1998·Environmental Science & Technology

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Citations

Jun 30, 2012·Analytical Chemistry·Payam AqaiMichel W F Nielen

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BETA
ELISA

Software Mentioned

ACDLabs

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