Immobilized Titanium (IV) Ion Affinity Chromatography Contributes to Efficient Proteomics Analysis of Cellular Nucleic Acid-Binding Proteins

Cellular nucleic acid-binding proteins (NABPs), namely, DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in many biological processes. However, extracting NABPs with high efficiency in living cells is challenging, which greatly limited their proteomics analysis and comprehensive characterization. Here, we discovered that titanium (IV) ion-immobilized metal affinity chromatography (Ti4+-IMAC) material could enrich DNA and RNA with high efficiency (96.82 ± 2.67 and 85.75 ± 2.99%, respectively). We therefore developed a Ti4+-IMAC method for the joint extraction of DBPs and RBPs. Through utilizing formaldehyde (FA) cross-linking, DBPs and RBPs were covalently linked to nucleic acids (NAs) and further denatured by organic solvents. After Ti4+-IMAC capture, 2000 proteins were identified in 293T cells, among which 417 DBPs and 999 RBPs were revealed, showing promising selectivity for NABPs. We further applied the Ti4+-IMAC capture method to lung cancer cell lines 95C and 95D, which have different tumor progression abilities. The DNA- and RNA-binding capabilities of many proteins have been dysregulated in 95D. Under our conditions, Ti4+-IMAC can be used as a selective and powerful tool for the comprehensive characterization of both DBPs and RBPs, which might be utilized to study their dynamic interactions with nucleic acids

Immobilized Titanium (IV) Ion Affinity Chromatography Contributes to Efficient Proteomics Analysis of Cellular Nucleic Acid-Binding Proteins

Cellular nucleic acid-binding proteins (NABPs), namely, DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in many biological processes. However, extracting NABPs with high efficiency in living cells is challenging, which greatly limited their proteomics analysis and comprehensive characterization. Here, we discovered that titanium (IV) ion-immobilized metal affinity chromatography (Ti4+-IMAC) material could enrich DNA and RNA with high efficiency (96.82 ± 2.67 and 85.75 ± 2.99%, respectively). We therefore developed a Ti4+-IMAC method for the joint extraction of DBPs and RBPs. Through utilizing formaldehyde (FA) cross-linking, DBPs and RBPs were covalently linked to nucleic acids (NAs) and further denatured by organic solvents. After Ti4+-IMAC capture, 2000 proteins were identified in 293T cells, among which 417 DBPs and 999 RBPs were revealed, showing promising selectivity for NABPs. We further applied the Ti4+-IMAC capture method to lung cancer cell lines 95C and 95D, which have different tumor progression abilities. The DNA- and RNA-binding capabilities of many proteins have been dysregulated in 95D. Under our conditions, Ti4+-IMAC can be used as a selective and powerful tool for the comprehensive characterization of both DBPs and RBPs, which might be utilized to study their dynamic interactions with nucleic acids

Immobilized Titanium (IV) Ion Affinity Chromatography Contributes to Efficient Proteomics Analysis of Cellular Nucleic Acid-Binding Proteins

Cellular nucleic acid-binding proteins (NABPs), namely, DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in many biological processes. However, extracting NABPs with high efficiency in living cells is challenging, which greatly limited their proteomics analysis and comprehensive characterization. Here, we discovered that titanium (IV) ion-immobilized metal affinity chromatography (Ti4+-IMAC) material could enrich DNA and RNA with high efficiency (96.82 ± 2.67 and 85.75 ± 2.99%, respectively). We therefore developed a Ti4+-IMAC method for the joint extraction of DBPs and RBPs. Through utilizing formaldehyde (FA) cross-linking, DBPs and RBPs were covalently linked to nucleic acids (NAs) and further denatured by organic solvents. After Ti4+-IMAC capture, 2000 proteins were identified in 293T cells, among which 417 DBPs and 999 RBPs were revealed, showing promising selectivity for NABPs. We further applied the Ti4+-IMAC capture method to lung cancer cell lines 95C and 95D, which have different tumor progression abilities. The DNA- and RNA-binding capabilities of many proteins have been dysregulated in 95D. Under our conditions, Ti4+-IMAC can be used as a selective and powerful tool for the comprehensive characterization of both DBPs and RBPs, which might be utilized to study their dynamic interactions with nucleic acids

Immobilized Titanium (IV) Ion Affinity Chromatography Contributes to Efficient Proteomics Analysis of Cellular Nucleic Acid-Binding Proteins

Cellular nucleic acid-binding proteins (NABPs), namely, DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in many biological processes. However, extracting NABPs with high efficiency in living cells is challenging, which greatly limited their proteomics analysis and comprehensive characterization. Here, we discovered that titanium (IV) ion-immobilized metal affinity chromatography (Ti4+-IMAC) material could enrich DNA and RNA with high efficiency (96.82 ± 2.67 and 85.75 ± 2.99%, respectively). We therefore developed a Ti4+-IMAC method for the joint extraction of DBPs and RBPs. Through utilizing formaldehyde (FA) cross-linking, DBPs and RBPs were covalently linked to nucleic acids (NAs) and further denatured by organic solvents. After Ti4+-IMAC capture, 2000 proteins were identified in 293T cells, among which 417 DBPs and 999 RBPs were revealed, showing promising selectivity for NABPs. We further applied the Ti4+-IMAC capture method to lung cancer cell lines 95C and 95D, which have different tumor progression abilities. The DNA- and RNA-binding capabilities of many proteins have been dysregulated in 95D. Under our conditions, Ti4+-IMAC can be used as a selective and powerful tool for the comprehensive characterization of both DBPs and RBPs, which might be utilized to study their dynamic interactions with nucleic acids

Immobilized Titanium (IV) Ion Affinity Chromatography Contributes to Efficient Proteomics Analysis of Cellular Nucleic Acid-Binding Proteins

Cellular nucleic acid-binding proteins (NABPs), namely, DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in many biological processes. However, extracting NABPs with high efficiency in living cells is challenging, which greatly limited their proteomics analysis and comprehensive characterization. Here, we discovered that titanium (IV) ion-immobilized metal affinity chromatography (Ti4+-IMAC) material could enrich DNA and RNA with high efficiency (96.82 ± 2.67 and 85.75 ± 2.99%, respectively). We therefore developed a Ti4+-IMAC method for the joint extraction of DBPs and RBPs. Through utilizing formaldehyde (FA) cross-linking, DBPs and RBPs were covalently linked to nucleic acids (NAs) and further denatured by organic solvents. After Ti4+-IMAC capture, 2000 proteins were identified in 293T cells, among which 417 DBPs and 999 RBPs were revealed, showing promising selectivity for NABPs. We further applied the Ti4+-IMAC capture method to lung cancer cell lines 95C and 95D, which have different tumor progression abilities. The DNA- and RNA-binding capabilities of many proteins have been dysregulated in 95D. Under our conditions, Ti4+-IMAC can be used as a selective and powerful tool for the comprehensive characterization of both DBPs and RBPs, which might be utilized to study their dynamic interactions with nucleic acids

Immobilized Titanium (IV) Ion Affinity Chromatography Contributes to Efficient Proteomics Analysis of Cellular Nucleic Acid-Binding Proteins

Cellular nucleic acid-binding proteins (NABPs), namely, DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in many biological processes. However, extracting NABPs with high efficiency in living cells is challenging, which greatly limited their proteomics analysis and comprehensive characterization. Here, we discovered that titanium (IV) ion-immobilized metal affinity chromatography (Ti4+-IMAC) material could enrich DNA and RNA with high efficiency (96.82 ± 2.67 and 85.75 ± 2.99%, respectively). We therefore developed a Ti4+-IMAC method for the joint extraction of DBPs and RBPs. Through utilizing formaldehyde (FA) cross-linking, DBPs and RBPs were covalently linked to nucleic acids (NAs) and further denatured by organic solvents. After Ti4+-IMAC capture, 2000 proteins were identified in 293T cells, among which 417 DBPs and 999 RBPs were revealed, showing promising selectivity for NABPs. We further applied the Ti4+-IMAC capture method to lung cancer cell lines 95C and 95D, which have different tumor progression abilities. The DNA- and RNA-binding capabilities of many proteins have been dysregulated in 95D. Under our conditions, Ti4+-IMAC can be used as a selective and powerful tool for the comprehensive characterization of both DBPs and RBPs, which might be utilized to study their dynamic interactions with nucleic acids

Synthesis of a Cationic Supramolecular Block Copolymer with Covalent and Noncovalent Polymer Blocks for Gene Delivery

The design and fabrication of safe and highly efficient nonviral vectors is the key scientific issue for the achievement of clinical gene therapy. Supramolecular cationic polymers have unique structures and specific functions compared to covalent cationic polymers, such as low cytotoxicity, excellent biodegradability, and smart environmental responsiveness, thereby showing great application prospect for gene therapy. However, supramolecular gene vectors are facile to be degraded under physiological conditions, leading to a significant reduction of gene transfection efficiency. In order to achieve highly efficient gene expression, it is necessary for supramolecular gene vectors being provided with appropriate biostability to overcome various cell obstacles. To this end, a novel cationic supramolecular block copolymer composed of a conventional polymer and a noncovalent polymer was constructed through robust β-cyclodextrin/ferrocene host–guest recognition. The resultant supramolecular block copolymer perfectly combines the advantages of both conventional polymers and supramolecular polymers ranging from structures to functions. This supramolecular copolymer not only has the ability to effectively condense pDNA for enhanced cell uptake, but also releases pDNA inside cancer cells triggered by H₂O₂, which can be utilized as a prospective nonviral delivery vehicle for gene delivery. The block polymer exhibited low cytotoxicity, good biostability, excellent biodegradability, and intelligent responsiveness, ascribing to the dynamic/reversible nature of noncovalent linkages. In vitro studies further illustrated that the supramolecular block polymer exhibited greatly improved gene transfection efficiency in cancer cells. This work offers an alternative platform for the exploitation of smart nonviral vehicles for specific cancer gene therapy in the future

In-Vial Temperature Gradient Headspace Single Drop Microextraction Designed by Multiphysics Simulation

Presented herein is a novel headspace single drop microextraction (HS-SDME) based on temperature gradient (TG) for an on-site preconcentration technique of volatile and semivolatile samples. First, an inner vial cap was designed as a cooling device for acceptor droplet in HS-SDME unit to achieve fast and efficient microextraction. Second, for the first time, an in-vial TG was generated between the donor phase in a sample vial at 80 °C and the acceptor droplet under the inner vial cap containing cooling liquid at −20 °C for a TG-HS-SDME. Third, a simple mathematic model and numerical simulations were developed by using heat transfer in fluids, Navier–Stokes and mass balance equations for conditional optimization, and dynamic illumination of the proposed extraction based on COMSOL Multiphysics. Five chlorophenols (CPs) were selected as model analytes to authenticate the proposed method. The comparisons revealed that the simulative results were in good agreement with the quantitative experiments, verifying the design of TG-HS-SDME via the numerical simulation. Under the optimum conditions, the extraction enrichments were improved from 302- to 388-fold within 2 min only, providing 3.5 to 4 times higher enrichment factors as compared to a typical HS-SDME. The simulation indicated that these improvements in the extraction kinetics could be attributed due to the applied temperature gap between the sample matrix and acceptor droplet within the small volume of headspace. Additionally, the experiments demonstrated a good linearity (0.03–100 μg/L, <i>R</i>² > 0.9986), low limit of detection (7–10 ng/L), and fair repeatability (<5.9% RSD, <i>n</i> = 6). All of the simulative and experimental results indicated the robustness, precision, and usefulness of TG-HS-SDME for trace analyses of analytes in a wide variety of environmental, pharmaceutical, food safety, and forensic samples