Use of Time-Resolved Fluorescence To Improve Sensitivity and Dynamic Range of Gel-Based Proteomics

Limitations in the sensitivity and dynamic range of two-dimensional gel electrophoresis (2-DE) are currently hampering its utility in global proteomics and biomarker discovery applications. In the current study, we present proof-of-concept analyses showing that introducing time-resolved fluorescence in the image acquisition step of in-gel protein quantification provides a sensitive and accurate method for subtracting confounding background fluorescence at the photon level. In-gel protein detection using the minimal difference gel electrophoresis workflow showed improvements in lowest limit of quantification in terms of CyDye molecules per pixel of 330-fold in the blue-green region (Cy2) and 8000-fold in the red region (Cy5) over conventional state-of-the-art image acquisition instrumentation, here represented by the Typhoon 9400 instrument. These improvements make possible the detection of low-abundance proteins present at sub-attomolar levels, thereby representing a quantum leap for the use of gel-based proteomics in biomarker discovery. These improvements were achieved using significantly lower laser powers and overall excitation times, thereby drastically decreasing photobleaching during repeated scanning. The single-fluorochrome detection limits achieved by the cumulative time-resolved emission two-dimensional electrophoresis (CuTEDGE) technology facilitates in-depth proteomics characterization of very scarce samples, for example, primary human tissue materials collected in clinical studies. The unique information provided by high-sensitivity 2-DE, including positional shifts due to post-translational modifications, may increase the chance to detect biomarker signatures of relevance for identification of disease subphenotypes

Efficient Condensation of DNA into Environmentally Responsive Polyplexes Produced from Block Catiomers Carrying Amine or Diamine Groups

The intracellular delivery of nucleic acids requires a vector system as they cannot diffuse across lipid membranes. Although polymeric transfecting agents have been extensively investigated, none of the proposed gene delivery vehicles fulfill all of the requirements needed for an effective therapy, namely, the ability to bind and compact DNA into polyplexes, stability in the serum environment, endosome-disrupting capacity, efficient intracellular DNA release, and low toxicity. The challenges are mainly attributed to conflicting properties such as stability vs efficient DNA release and toxicity vs efficient endosome-disrupting capacity. Accordingly, investigations aimed at safe and efficient therapies are still essential to achieving gene therapy clinical success. Taking into account the mentioned issues, herein we have evaluated the DNA condensation ability of poly­(ethylene oxide)₁₁₃-<i>b</i>-poly­[2-(di­iso­propyl­amino)­ethyl meth­acrylate]₅₀ (PEO₁₁₃-<i>b</i>-PDPA₅₀), poly­(ethylene oxide)₁₁₃-<i>b</i>-poly­[2-(di­ethyl­amino)­ethyl meth­acrylate]₅₀ (PEO₁₁₃-<i>b</i>-PDEA₅₀), poly­[oligo­(ethylene glycol)­methyl ether meth­acrylate]₇₀-<i>b</i>-poly­[oligo­(ethylene glycol)­methyl ether meth­acrylate₁₀-<i>co</i>-2-­(diethylamino)­ethyl meth­acrylate₄₇-<i>co</i>-2-(diisopropylamino)­ethyl meth­acrylate₄₇] (POEGMA₇₀-<i>b</i>-P­(OEGMA₁₀-<i>co</i>-DEA₄₇-<i>co</i>-DPA₄₇), and poly­[oligo­(ethylene glycol)­methyl ether meth­acrylate]₇₀-<i>b</i>-poly­{oligo­(ethylene glycol)­methyl ether meth­acrylate₁₀-<i>co</i>-2-methyl­acrylic acid 2-[(2-(di­methyl­amino)­ethyl)­methyl­amino]­ethyl ester₄₄} (POEGMA₇₀-<i>b</i>-P­(OEGMA₁₀-<i>co</i>-DAMA₄₄). Block copolymers PEO₁₁₃-<i>b</i>-PDEA₅₀ and POEGMA₇₀-<i>b</i>-P­(OEGMA₁₀-<i>co</i>-DEA₄₇-<i>co</i>-DPA₄₇) were evidenced to properly condense DNA into particles with a desirable size for cellular uptake via endocytic pathways (<i>R</i>_H ≈ 65–85 nm). The structure of the polyplexes was characterized in detail by scattering techniques and atomic force microscopy. The isothermal titration calorimetric data revealed that the polymer/DNA binding is endothermic; therefore, the process in entropically driven. The combination of results supports that POEGMA₇₀-<i>b</i>-P­(OEGMA₁₀-<i>co</i>-DEA₄₇-<i>co</i>-DPA₄₇) condenses DNA more efficiently and with higher thermodynamic outputs than does PEO₁₁₃-<i>b</i>-PDEA₅₀. Finally, circular dichroism spectroscopy indicated that the conformation of DNA remained the same after complexation and that the polyplexes are very stable in the serum environment