title: Improved silicon nanowire field-effect transistors for fast protein-protein interaction screening.
abstract: 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 Ca(2+)-sensing protein that is ubiquitously expressed in cells and its interaction with target molecules is Ca(2+)-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 Ca(2+)-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 Ca(2+)/CaM-dependent protein kinase IIα 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-CalnI-modified SiNW-FET. The proteins that interacted with CalnI were eluted and verified by MS. The Ca(2+)-dependent interaction between GST-CalnI and one of the candidates, heat shock protein 70, was re-confirmed via the SiNW-FET measurement. Our results demonstrate the effectiveness of combining MS with SiNW-FETs to quickly screen interacting molecules from cell lysates.
authors.affiliation: Institute of Zoology, National Taiwan University, Taipei, Taiwan.
authors.forename: Ti-Yu
authors.initials: TY
authors.lastname: Lin
authors.forename: Bor-Ran
authors.initials: BR
authors.lastname: Li
authors.forename: Sheng-Ta
authors.initials: ST
authors.lastname: Tsai
authors.forename: Chien-Wei
authors.initials: CW
authors.lastname: Chen
authors.forename: Chung-Hsuan
authors.initials: CH
authors.lastname: Chen
authors.forename: Yit-Tsong
authors.initials: YT
authors.lastname: Chen
authors.forename: Chien-Yuan
authors.initials: CY
authors.lastname: Pan
mesh_terms: D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry*; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation; D053770:Nanowires / Q000737:chemistry*; D011485:Protein Binding; D012825:Silicon / Q000737:chemistry*; D014173:Transistors, Electronic
chemical_list: D002147:Calmodulin; D005982:Glutathione Transferase; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D005978:Glutathione; D012825:Silicon