Exploring the interactome: microfluidic isolation of proteins and interacting partners for quantitative analysis by electron microscopy.

Multimolecular protein complexes are important for many cellular processes. However, the stochastic nature of the cellular interactome makes the experimental detection of complex protein assemblies difficult and quantitative analysis at the single molecule level essential. Here, we present a fast and simple microfluidic method for (i) the quantitative isolation of endogenous levels of untagged protein complexes from minute volumes of cell lysates under close to physiological conditions and (ii) the labeling of specific components constituting these complexes. The method presented uses specific antibodies that are conjugated via a photocleavable linker to magnetic beads that are trapped in microcapillaries to immobilize the target proteins. Proteins are released by photocleavage, eluted, and subsequently analyzed by quantitative transmission electron microscopy at the single molecule level. Additionally, before photocleavage, immunogold can be employed to label proteins that interact with the primary target protein. Thus, the presented method provides a new way to study the interactome and, in combination with single molecule transmission electron microscopy, to structurally characterize the large, dynamic, heterogeneous multimolecular protein complexes formed.

Single-cell lysis for visual analysis by electron microscopy.

The stochastic nature of biological systems makes the study of individual cells a necessity in systems biology. Yet, handling and disruption of single cells and the analysis of the relatively low concentrations of their protein components still challenges available techniques. Transmission electron microscopy (TEM) allows for the analysis of proteins at the single-molecule level. Here, we present a system for single-cell lysis under light microscopy observation, followed by rapid uptake of the cell lysate. Eukaryotic cells were grown on conductively coated glass slides and observed by light microscopy. A custom-designed microcapillary electrode was used to target and lyse individual cells with electrical pulses. Nanoliter volumes were subsequently aspirated into the microcapillary and dispensed onto an electron microscopy grid for TEM inspection. We show, that the cell lysis and preparation method conserves protein structures well and is suitable for visual analysis by TEM.

Ionic liquids as matrices in microfluidic sample deposition for high-mass matrix- assisted laser desorption/ionization mass spectrometry.

Sample preparation for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) via a microfluidic deposition device using ionic liquid matrices addresses several problems of standard protocols with crystalline matrices, such as the heterogeneity of sample spots due to the co-crystallization of sample and matrix and the limited capability for high-throughput analysis. Since ionic liquid matrices do not solidify during the measurement, the resulting sample spots are homogeneous. The use of these matrices is also beneficial for automated sample preparation, since crystallization of the matrix is avoided and, thus, no clogging of the spotting device can occur. The applicability of ionic liquids to the analysis of biomolecules with high molecular weights, up to ≈ 1 MDa is shown, as well as a good sensitivity (5 fmol) for recombinant human fibronectin, a protein with a molecular weight of 226 kDa. Microfluidic sample deposition of proteins with high molecular weights will, in the future, allow parallel sample preparation for MALDI-MS and for electron microscopy.

Connecting µ-fluidics to electron microscopy.

A versatile methodology for electron microscopy (EM) grid preparation enabling total content sample analysis is presented. A microfluidic-dialysis conditioning module to desalt or mix samples with negative stain solution is used, combined with a robotic writing table to micro-pattern the EM grids. The method allows heterogeneous samples of minute volumes to be processed at physiological pH for structure and mass analysis, and allows the preparation characteristics to be finely tuned.