Microfluidic Tissue Mesodissection in Molecular Cancer Diagnostics.

We present a mesodissection platform that retains the advantages of laser-based dissection instrumentation with the speed and ease of manual dissection. Tissue dissection in clinical laboratories is often performed by manually scraping a physician-selected region from standard glass slide mounts. In this manner, costs associated with dissection remain low, but spatial resolution is compromised. In contrast, laser microdissection methods maintain spatial resolution that matches the requirements for analysis of important tissue heterogeneity but remains costly and labor intensive. We demonstrate a microfluidic tool for rapid extraction of histological regions of interest from formalin-fixed paraffin-embedded tissue, which uses a simple and automated method that is compatible with most downstream enzymatic reactions, including protocols used for next-generation DNA sequencing.

Rapid matrix-assisted refolding of histidine-tagged proteins.

The formation of inclusion bodies (IBs)--amorphous aggregates of misfolded insoluble protein--during recombinant protein expression, is still one of the biggest bottlenecks in protein science. We have developed and analyzed a rapid parallel approach for matrix-assisted refolding of recombinant His(6)-tagged proteins. Efficiencies of matrix-assisted refolding were screened in a 96-well format. The developed methodology allowed the efficient refolding of five different test proteins, including monomeric and oligomeric proteins. Compared to refolding in-solution, the matrix-assisted refolding strategy proved equal or better for all five proteins tested. Interestingly, specifically oligomeric proteins displayed significantly higher levels of refolding compared to refolding in-solution. Mechanistically, matrix-assisted folding seems to differ from folding in-solution, as the reaction proceeds more rapidly and shows a remarkably different concentration dependence--it allows refolding at up to 1000-fold higher protein concentration than folding in-solution.

Structure and dynamics of dark-state bovine rhodopsin revealed by chemical cross-linking and high-resolution mass spectrometry.

Recent work using chemical cross-linking to define interresidue distance constraints in proteins has shown that these constraints are useful for testing tertiary structural models. We applied this approach to the G-protein-coupled receptor bovine rhodopsin in its native membrane using lysine- and cysteine-targeted bifunctional cross-linking reagents. Cross-linked proteolytic peptides of rhodopsin were identified by combined liquid chromatography and FT-ICR mass spectrometry with automated data-reduction and assignment software. Tandem mass spectrometry was used to verify cross-link assignments and locate the exact sites of cross-link attachment. Cross-links were observed to form between 10 pairs of residues in dark-state rhodopsin. For each pair, cross-linkers with a range of linker lengths were tested to determine an experimental distance-of-closest-approach (DCA) between reactive side-chain atoms. In all, 28 cross-links were identified using seven different cross-linking reagents. Molecular mechanics procedures were applied to published crystal structure data to calculate energetically achievable theoretical DCAs between reactive atoms without altering the position of the protein backbone. Experimentally measured DCAs are generally in good agreement with the theoretical DCAs. However, a cross-link between C316 and K325 in the C-terminal region cannot be rationalized by DCA simulations and suggests that backbone reorientation relative to the crystal coordinates occurs on the timescale of cross-linking reactions. Biochemical and spectroscopic data from other studies have found that the C-terminal region is highly mobile in solution and not fully represented by X-ray crystallography data. Our results show that chemical cross-linking can provide reliable three-dimensional structural information and insight into local conformational dynamics in a membrane protein.

A novel protein crosslinking reagent for the determination of moderate resolution protein structures by mass spectrometry (MS3-D).

A new approach to the determination of moderate resolution protein structures, termed MS3-D-Mass Spectrometry in 3 Dimensions-has recently been disclosed. The method involves the formation of covalent crosslinks between reactive residues on the protein surface, the determination of the location of those crosslinks in primary sequence space by mass spectrometry, and then the imposition of a distance constraint upon the location of the respective side chains during distance geometry calculations of protein structure. MS3-D is rapid, requires small amounts of protein, and works in native biochemical conditions. Therefore, it offers the potential for determination of the structures of all proteins expressed by an organism in a high throughput manner. However, the methodology is completely dependent upon the production of chemical crosslinks and technical limitations of available crosslinkers have proven problematic in generalization and automation of the method for the determination of the structures of complete proteomes. Presented herein is the design, synthesis, and proofing of a novel modular protein crosslinking reagent designed to enhance hydrophilicity, provide an increased effective signal to noise ratio for MS3-D, and allow the sampling of a wider variety of side chains during the process.

Improved assembly of multimeric genes for the biosynthetic production of protein polymers.

We report a general method for the construction of highly repetitive synthetic genes and their use in the biosynthetic production of artificial protein polymers. Through the application of improved recombinant DNA techniques and high-throughput screening methods, we have developed a facile approach to rapid gene assembly and cloning which is widely applicable in the biosynthesis of novel protein polymers. Using this technique, synthetic genes encoding tandem repeats of the beta-sheet forming amino acid sequence AEAEAKAK were constructed and subsequently cloned into a bacterial expression host for inducible protein production. A 17-kDa fusion protein, poly-EAK9, was isolated from Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. The amino acid sequence and molecular weight were confirmed by amino acid analysis, N-terminal sequencing, and MALDI-TOF mass spectrometry. Circular dichroism studies on the artificial protein poly-EAK9 demonstrate the formation of a beta-sheet structure in aqueous solution.