Huimin Bao

Immobilization of trypsin in polyaniline-coated nano-Fe3O4/carbon nanotube composite for protein digestion.

In this report, a four-component nanocomposite, trypsin-immobilized polyaniline-coated Fe(3)O(4)/carbon nanotube composite, was synthesized for highly efficient protein digestion. Fe(3)O(4) was deposited by the chemical coprecipitation of Fe(2+) and Fe(3+) in an alkaline solution containing carbon nanotubes (CNTs) to prepare nano-Fe(3)O(4)/CNT composite. Subsequently, polyaniline (PA) was assembled on the Fe(3)O(4)/CNT composite by the in situ polymerization of aniline in the presence of trypsin to obtain trypsin-immobilized PA/Fe(3)O(4)/CNT nanocomposite. The novel 1D superparamagnetic biomaterial has been characterized by TEM, SEM, XRD, and magnetometric analysis. The feasibility and performance of the unique magnetic biomaterial have been demonstrated by the tryptic digestion of bovine serum albumin, myoglobin, and lysozyme within 5min. The digests were identified by MALDI-TOF MS with sequence coverages that were comparable to those obtained from the conventional in-solution tryptic digestion. The present biocomposite offers considerable promise for protein analysis due to its high magnetic responsivity and excellent dispersibility. It can be easily isolated from the digests with the aid of an external magnetic field. Because the enzyme-immobilized nanocomposite can be prepared by a simple two-step deposition approach at low cost, it may find a wide range of biological applications including proteome research.

Infrared-Assisted On-Plate Proteolysis for MALDI-TOF-MS Peptide Mapping

In this report, infrared (IR)-assisted on-plate proteolysis has been developed for rapid peptide mapping. Protein solutions containing trypsin were allowed to digest directly on the spots of matrix-assisted laser desorption/ionization (MALDI) plates under IR radiation. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of bovine serum albumin (BSA) and cytochrome c (Cyt-c). It was demonstrated that IR radiation substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min. The digests were identified by MALDI time-of-flight mass spectrometry with sequence coverages of 55 (BSA) and 75% (Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion. The suitability of IR-assisted on-plate proteolysis to complex proteins was demonstrated by digesting human serum and casein extracted from commercially available milk sample. The present proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.

Far‐Infrared‐Assisted Preparation of a Graphene–Nickel Nanoparticle Hybrid for the Enrichment of Proteins and Peptides

A new approach based on far infrared-assisted in situ reduction was developed for the facile one-step preparation of graphene-nickel nanoparticle hybrid by refluxing a mixture solution containing graphene oxide, nickel(II) sulfate, and hydrazine over an far-infrared heater. The reduction time was as short as 20 min. The structure of the material was investigated by transmission electron microscopy, scanning electron microscopy, X-ray diffraction, energy dispersive spectroscopy, vibrating sample magnetometery, and Fourier transform infrared spectroscopy. Magnetic investigations indicate that the grapheme-nickel nanoparticle hybrid exhibits ferromagnetic behavior at room temperature. Meanwhile, the hybrid was successfully employed in the enrichment and identification of proteins and peptides in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based on its excellent magnetic responsibility, high dispersibility, large surface area, and hydrophobicity, indicating great promise for a wide range of applications.

Proteomic dissection of cell type-specific H2AX-interacting protein complex associated with hepatocellular carcinoma.

The replacement histone variant H2AX senses DNA double-strand breaks (DSBs) and recruits characteristic sets of proteins at its phosphorylated (gamma-H2AX) foci for concurrent DNA repair. We reasoned that the H2AX interaction network, or interactome, formed in the tumor-associated DNA DSB environment such as in hepatocellular carcinoma (HCC) cells, where preneoplastic lesions frequently occur, is indicative of HCC pathogenic status. By using an in vivo dual-tagging quantitative proteomic method, we identified 102 H2AX-specific interacting partners in HCC cells that stably expressed FLAG-tagged H2AX at close to the endogenous level. Using bioinformatics tools for data-dependent network analysis, we further found binary relationships among these interactors in defined pathway modules, implicating H2AX in a multifunctional role of coordinating a variety of biological pathways involved in DNA damage recognition and DNA repair, apoptosis, nucleic acid metabolism, Ca(2+)-binding signaling, cell cycle, etc. Furthermore, our observations suggest that these pathways interconnect through key pathway components or H2AX interactors. The physiological accuracy of our quantitative proteomic approach in determining H2AX-specific interactors was evaluated by both coimmunoprecipitation/ immunoblotting and confocal colocalization experiments performed on HCC cells. Due to their involvement in diverse functions, the H2AX interactors involved in different pathway modules, such as Poly(ADP-ribose) polymerase 1, 14-3-3 zeta, coflin 1, and peflin 1, were examined for their relative H2AX binding affinities in paired hepatocytes and HCC cells. Treatment with the DSB-inducing agent bleomycin enhanced binding of these proteins to H2AX, suggesting an active role of H2AX in coordinating the functional pathways of each protein in DNA damage recognition and repair.

Determination of the rate constants and activation energy of acetaminophen hydrolysis by capillary electrophoresis.

A method based on capillary electrophoresis with electrochemical detection (CE-ED) was developed for the simultaneous determination of p-aminophenol and acetaminophen in the hydrolysates of acetaminophen. Effects of several important factors such as the acidity and concentration of running buffer, separation voltage, injection time, and working potential were investigated to acquire the optimum conditions. The detection electrode was a 300 microm carbon disc electrode at a working potential of +0.80 V (versus SCE). The two analytes can be well separated within 6 min in a 50 cm length fused silica capillary at a separation voltage of 18 kV in a 25 mM phosphate buffer (pH 6.5). The rate constants of acetaminophen hydrolysis in 0.5 M HCl at different temperatures were determined by monitoring the concentration changes of acetaminophen. At 70, 80, 90 and 100 degrees C, the measured rate constants of acetaminophen hydrolysis were 5.027 x 10(-3), 8.522 x 10(-3), 18.60 x 10(-3) and 32.76 x 10(-3) min(-1), respectively. The activation energy for acetaminophen hydrolysis was calculated to be 68.13 kJ mol(-1), which is in good agreement with the value in the literature.

Efficient in-gel proteolysis accelerated by infrared radiation for protein identification

In this report, infrared (IR) radiation was employed to enhance the efficiency of in-gel proteolysis for MS-based protein identification. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the target protein bands excised from polyacrylamide gel were cut into small pieces that were further treated in trypsin solution. Subsequently, the wet gel pieces sealed in transparent Eppendorf tubes were exposed to an IR lamp to perform IR-assisted in-gel digestion. To demonstrate the feasibility and performance of the novel digestion approach, it was employed to digest BSA and cytochrome c (Cyt-c) in polyacrylamide gels after SDS-PAGE separations. The results indicated that IR radiation substantially enhanced the efficiency of in-gel proteolysis and the digestion time was significantly reduced to 5 min compared to 16 h for conventional in-gel digestion. The obtained digests were further identified by MALDI-TOF MS with improved sequence coverages. The suitability of IR-assisted in-gel proteolysis to real protein samples was demonstrated by digesting and identifying human serum albumin in gel separated from human serum by SDS-PAGE. The present proteolysis strategy is simple and efficient, offering great promise for the high-throughput protein identification in proteomics research.

Infrared-assisted proteolysis using trypsin-immobilized silica microspheres for peptide mapping.

A novel proteolysis approach was developed by using infrared (IR) radiation and trypsin‐immobilized silica microspheres. Protein solutions containing trypsin‐immobilized microspheres in sealed transparent Eppendorf tubes were allowed to digest under an IR lamp at 37°C. The feasibility and performance of the present proteolysis approach were demonstrated by the digestion of BSA and cytochrome c (Cyt‐c) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI‐TOF‐MS with the sequence coverages of 54% (BSA) and 83% (Cyt‐c) that were better than those obtained by conventional in‐solution tryptic digestion. The suitability of the new digestion approach to complex proteins was demonstrated by digesting human serum. The present proteolysis strategy is simple and efficient and will find a wide range of application in protein identification.

Immobilization of trypsin via graphene oxide-silica composite for efficient microchip proteolysis.

In this report, trypsin was covalently immobilized in the graphene oxide (GO)-silica composite coating on the channel wall of poly(methyl methacrylate) (PMMA) microchips to fabricate microfluidic bioreactors for highly efficient proteolysis. A mixture solution containing GO nanosheets and silica gel was injected into the channels to form coating. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were used as carboxyl activating agents to crosslink the primary amino groups of trypsin to the carboxyl groups of the entrapped GO sheets in the composite to realize covalent immobilization. The feasibility and performance of the novel GO-based microfluidic bioreactors were demonstrated by the digestion of hemoglobin (HEM), cytochrome c (Cyt-c), myoglobin (MYO), and ovalbumin (OVA) and the digestion time was significantly reduced to 5s. The obtained digests were identified by MALDI-TOF MS with the sequence coverages of 95%, 76%, 69%, and 55% for HEM, Cyt-c, MYO, and OVA, respectively. The suitability of the prepared bioreactors to complex proteins was demonstrated by digesting human serum.

Immobilization of trypsin on poly(urea-formaldehyde)-coated fiberglass cores in microchip for highly efficient proteolysis.

Trypsin was covalently immobilized on poly(urea‐formaldehyde)‐coated fiberglass cores based on the condensation reaction between poly(urea‐formaldehyde) and trypsin for efficient microfluidic proteolysis in this work. Prior to use, a piece of the trypsin‐immobilized fiber was inserted into the main channel of a microchip under a magnifier to form a core‐changeable bioreactor. Because trypsin was not permanently immobilized on the channel wall, the novel bioreactor was regenerable. Two standard proteins, hemoglobin (HEM) and lysozyme (LYS), were digested by the unique bioreactor to demonstrate its feasibility and performance. The interaction time between the flowing proteins and the immobilized trypsin was evaluated to be less than 10 s. The peptides in the digests were identified by MALDI‐TOF MS to obtain PMF. The results indicated that digestion performance of the microfluidic bioreactor was better than that of 12‐h in‐solution digestion.

Accelerated proteolysis in alternating electric fields for peptide mapping

Sinusoidal alternating voltages (typically 5 V) were employed to enhance the efficiency of proteolysis for peptide mapping in this work. Protein solutions containing trypsin were allowed to digest with the assistance of alternating electric fields (AEFs) between a pair of platinum wire electrodes in Eppendorf tubes. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of several standard proteins. It was demonstrated that AEFs significantly accelerated in-solution proteolysis and the digestion time was substantially reduced to 5 min. The digests were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with sequence coverages that were comparable to those obtained by using conventional 12-h in-solution proteolysis. The suitability of AEF-assisted proteolysis to real protein samples was demonstrated by digesting and identifying human serum albumin in gel separated from human serum by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). The present proteolysis strategy is simple and efficient and will find a wide range of applications in protein identification.