Fabrication of a monolithic, macroporous diallyl maleate-based material and its application for fast separation of intact proteins from human plasma with reversed-phase chromatography.

A monolithic diallyl maleate-based column was developed and used to separate intact proteins from complex bio-samples with high-performance liquid chromatography. The resulting monolith exhibited a relatively uniform porous structure with macro-pores. A sample of standard proteins mixture was used to explore the reversed-phase mechanism for fast separation on the homemade monolithic column. The observed retention time increased with the values of the diameters, relative hydrophobicities and molecular weights of the standard proteins, which mainly depends on the relative hydrophobicity of the proteins. It is worth mentioning that the mask of the three most abundant proteins in human plasma can be avoided, thus providing an opportunity for the middle- and low-abundance proteins to be detected. According to this exploration, the fast separation of human plasma proteins on the diallyl maleate-based monolithic column was achieved in 15 min. The chromatographic fractions were identified by liquid chromatography-tandem mass spectrometry, and the results indicate that the present method is an outstanding method for the fast and efficient fractionation of human plasma that will be significant sense for plasma proteomics research, especially for exploring new disease marker and drug target.

In situ synthesis of a monolithic material with multi-sized pores and its chromatographic properties for the separation of intact proteins from human plasma.

The fabrication of a monolithic allyl phenoxyacetate-based material was proposed via the in situ radical polymerization using ethylene dimethacrylate as the crosslinker and 2,2'-azobisisobutyronitrile as the initiator within a stainless steel column (50 mm × 4.6 mm i.d.). The effects of the porogen composition, the crosslinker amount and the monomer type on the resulting monoliths were investigated. The morphology of the monoliths was characterized using scanning electron microscopy and a nitrogen adsorption-desorption instrument, and the pore structure was characterized using mercury intrusion porosimetry. The results indicate that the optimized monolith has a micro-, meso- and macro- multi-sized pore structure with a high specific surface area of 260.66 m2 g-1. The resulting monoliths were used as stationary phases for the separation of proteins from bio-samples, including a mixture of six standard proteins, chicken egg whites, snailase and human plasma, using high-performance liquid chromatography. Compared to optimized glycidyl methacrylate-based and styrene-based monolithic columns, the allyl phenoxyacetate-based monolithic column exhibited improved selectivity in the separation of proteins. Furthermore, the present method avoids the masking of highly abundant proteins, such as human serum albumin, immunoglobulin G and human fibrinogen, in the detection of middle- or low-abundance proteins in human plasma. The protein identification results obtained from liquid chromatography/mass spectrometry indicate that the present method is an outstanding method for efficient fractionation of human plasma, which will be especially useful in future plasma proteomics research.

Preparation of a hydroxyethyl-based monolithic column and its application in the isolation of intact proteins from complex bio-samples.

In this work, a monolithic hydroxyethyl-based column was fabricated in a stainless-steel column (50 mm × 4.6 mm i.d.) via radical polymerization technique using hydroxyethyl methylacrylate as the monomer. The morphology and pore size distribution indicate that the optimized monolith has a relatively uniform structure with macro-pores. The homemade monolith was used as the stationary phase of high performance liquid chromatography for the separation of intact proteins from complex bio-samples, including human plasma, egg white and snailase. The resulting monolith shows excellent selectivity for intact proteins mainly depending on the different relative hydrophobicity of the objective proteins with reversed-phase liquid chromatographic mechanism. Besides, the hydrogen-bond interaction and electrostatic interaction were the additional interactions in the chromatographic separation owing to hydroxyl groups present in the surface of monolithic material.

A novel poly (NMA-co-DEA-co-EDMA) monolithic column as a sorbent for online solid-phase extraction and its application in the determination of ß-sitosterol in plant oil samples.

A novel monolithic column was prepared by in-situ free radical polymerization using N-methylolacrylamide (NMA) and N,N-diethylacrylamide (DEA) as co-monomers. The monolith was characterized by scanning electron microscopy (SEM) and its nitrogen adsorption-desorption isotherm, and it was used as a solid phase extraction (SPE) absorbent for the online enrichment of ß-sitosterol by high performance liquid chromatography. The optimized method had good linearity, and the linear regression coefficient was 0.998. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.006 mg/mL and 0.02 mg/mL, respectively. The interday and intraday accuracies were less than 7.28%. The spiked recoveries of ß-sitosterol in the six plant oil were 90.96-103.56%. The maximum amount of ß-sitosterol adsorbed on the monolithic column was 12.69 mg/g, and the enrichment factor of ß-sitosterol was 78. The results showed that the monolith could be used as an online SPE absorbent for the determination of ß-sitosterol in plant oil samples.

Separation of proteins from complex bio-matrix samples using a double-functionalized polymer monolithic column.

A double-functionalized polymer monolithic column was fabricated within the confines of a stainless-steel column (50 mm × 4.6 mm i.d.) via a facile method using iron porphyrin, ionic liquid (1-allyl-3-methylimidazolium chloride) and 1,10-decanediol dimethacrylate as tri-monomers; ethylene dimethacrylate as a crosslinker; polyethylene glycol 400 and N,N-dimethylformamide as co-porogens; benzoyl peroxide and N,N-dimethyl aniline as the redox initiation system. Results obtained from scanning electron microscopy, nitrogen adsorption-desorption, and mercury intrusion porosimetry confirmed the uniform pore structure and the pore size distribution of macro-pores. The home-made monolith was further characterized by elemental analysis to investigate the elemental composition of Fe supplied by iron porphyrin, confirming the synthetic process. The resulting optimized monolithic column was used as the stationary phase in high performance liquid chromatography for separating proteins, such as mixture of standard proteins, egg white, and human plasma, exhibiting good selectivity and high performance. It is worth noting that the home-made double-functionalized polymer monolithic column shows excellent selectivity for fractionation separation of human plasma proteins, and it is a promising separation tool for complex bio-samples in proteomic research.

Fractionation separation of human plasma proteins using HPLC with a homemade iron porphyrin based monolithic column.

In this work a polymer monolithic column was fabricated within the confines of a stainless steel column (50×4.6mm i.d.) via radical polymerization by using iron porphyrin and butyl methacrylate as co-monomers, ethylene glycol dimethacrylate as crosslinking agent, ethylene glycol, isopropyl alcohol and N, N-dimethylformamide as tri-porogens, benzoyl peroxide and N,N-dimethylaniline as initiators. The resulting monolithic column was characterized by elemental analysis, scanning electron microscopy, nitrogen adsorption BET surface area, and mercury intrusion porosimetry, respectively. Results showed that the homemade monolith occupied relatively uniform pore structure, low back pressure, and enhanced selectivity for proteins in complex bio-samples. The present work described a simple and efficient method for "fractionation separation" of human plasma proteins, and it is a promising separation method for complex bio-samples in proteomic research.

The Ac124 protein is not essential for the propagation of Autographa californica multiple nucleopolyhedrovirus, but it is a viral pathogenicity factor.

orf124 (ac124) of AcMNPV is one of the highly conserved unique genes in group I lepidopteran nucleopolyhedroviruses. So far, its function remains unknown. In this study, infection with a virus expressing an ac124-gfp fusion showed that Ac124 localized to the cytoplasm throughout the infection. In addition, an ac124 knockout virus was generated to determine the role of ac124 in the baculovirus life cycle. Our results showed that an ac124 knockout AcMNPV could produce infectious budded viruses (BVs) and occlusion bodies (OBs) like those produced by the wild virus and ac124 repair virus. These three viruses had similar growth kinetics during the infection phase. There was no significant difference in nucleocapsids, occlusion-derived viruses and OBs visualized by electron microscopy. The ac124 deletion mutant did not reduce AcMNPV infectivity for S. exigua in an LD50 bioassay. However, it took 20 h longer for the ac124 deletion mutant to kill S. exigua than wild-type virus in the LT50 bioassay. Altogether, these results demonstrate that ac124 is not required for viral replication, but it accelerates the killing of infected larvae.

Autographa californica multiple nucleopolyhedrovirus PK-1 is essential for nucleocapsid assembly.

PK-1 (Ac10) is a baculovirus-encoded serine/threonine kinase and its function is unclear. Our results showed that a pk-1 knockout AcMNPV failed to produce infectious progeny, while the pk-1 repair virus could rescue this defect. qPCR analysis demonstrated that pk-1 deletion did not affect viral DNA replication. Analysis of the repaired recombinants with truncated pk-1 mutants demonstrated that the catalytic domain of protein kinases of PK-1 was essential to viral infectivity. Moreover, those PK-1 mutants that could rescue the infectious BV production defect exhibited kinase activity in vitro. Therefore, it is suggested that the kinase activity of PK-1 is essential in regulating viral propagation. Electron microscopy revealed that pk-1 deletion affected the formation of normal nucleocapsids. Masses of electron-lucent tubular structures were present in cell transfected with pk-1 knockout bacmid. Therefore, PK-1 appears to phosphorylate some viral or cellular proteins that are essential for DNA packaging to regulate nucleocapsid assembly.