Ambient nanoelectrospray ionization with in-line microdialysis for spatially resolved transient biochemical monitoring within cell culture environments.

We have developed a new mass spectrometry (MS) based approach for continuous, spatially resolved in vitro biochemical detection and demonstrated its utility in a 3-D cell culture system. Extracellular liquid is passively extracted at a low flow rate (~10 nL/s) through a small bore silica capillary (ID 50 μm); inline microdialysis (MD) removes ions that would interfere with mass spectrometric analysis, and the sample is ionized by nanoelectrospray ionization (nano-ESI) and mass analyzed in a time-of-flight mass spectrometer. The system successfully detects low-volume, low-concentration releases of a small protein (8 μL of 5 μM cytochrome-c, molecular mass ~12 kDa) and exhibits ~1 min temporal resolution.

Cell-free expression of soluble and membrane proteins in an array device for drug screening.

Enzymes and membrane protein receptors represent almost three-quarters of all current drug targets. As a result, it would be beneficial to have a platform to produce them in a high-throughput format for drug screening. We have developed a miniaturized fluid array device for cell-free protein synthesis, and the device was exploited to produce both soluble and membrane proteins.

Miniaturized fluid array for high-throughput protein expression.

We describe a miniaturized fluid array device for high-throughput cell-free protein synthesis (CFPS), aiming to match the throughput and scale of gene discovery. Current practice of using E. coli cells for production of recombinant proteins is difficult and cost-prohibitive to implement in a high-throughput format.