中央研究院 原子與分子科學研究所

Understanding how proteins interact with each other is the basis for studying the biological mechanisms behind various physiological activities. Silicon nanowire field-effect transistors (SiNW-FETs) are sensitive sensors used to detect biomolecular interactions in real-time. However, the majority of the applications that use SiNW-FETs are for known interactions between different molecules. To explore the capability of SiNW-FETs as fast screening devices to identify unknown interacting molecules, we applied mass spectrometry (MS) to analyze molecules reversibly bound to the SiNW-FETs. Calmodulin (CaM) is a Ca²⁺- sensing protein that is ubiquitously expressed in cells and its interaction with target molecules is Ca²⁺- dependent. By modifying the SiNW-FET surface with glutathione, glutathione S-transferase (GST)-tagged CaM binds reversibly to the SiNW-FET. We first verified the Ca²⁺-dependent interaction between GST–CaM and purified troponin I, which is involved in muscle contraction, through the conductance changes of the SiNW-FET. Furthermore, the cell lysate containing overexpressed Ca²⁺/CaM-dependent protein kinase IIa induced a conductance change in the GST–CaM-modified SiNW-FET. The bound proteins were eluted and subsequently identified by MS as CaM and kinase. In another example, candidate proteins from neuronal cell lysates interacting with calneuron I (CalnI), a CaM-like protein, were captured with a GST–CalnImodified SiNW-FET. The proteins that interacted with CalnI were eluted and verified by MS. The Ca²⁺- dependent interaction between GST–CalnI and one of the candidates, heat shock protein 70, was reconfirmed via the SiNW-FET measurement. Our results demonstrate the effectiveness of combining MS with SiNW-FETs to quickly screen interacting molecules from cell lysates.