Functionalized PEG‐b‐PAGE‐b‐PLGA triblock terpolymers as materials for nanoparticle preparation

A series of poly(ethylene glycol)‐block‐poly(allyl glycidyl ether) (PEG‐b‐PAGE) macroinitiators are prepared using the living anionic ring‐opening polymerization (AROP) technique, and applied for further copolymerization studies. To overcome the low reactivity of the secondary hydroxyl end‐group of the PAGE block, a primary hydroxyl group is introduced into the macroinitiators via trityl and tert‐butyl‐dimethylsilane protective groups. The modified macroinitiators are used for copolymerization by applying different amounts of PEG‐b‐PAGE (5, 10, and 15%) and different PLGA lengths. To study their properties, nanoparticles from selected polymers are prepared and characterized by dynamic light scattering and scanning electron microscopy showing spherical particles with diameters around 200 nm and low PDI_particle values of 0.03–0.1. An advantage of the obtained polymers is the presence of double bonds in the side chain, which enables the modification via, for example, thiol‐ene reactions. For this purpose tertiary 2‐(dimethylamino)ethanethiol), acetylated thiogalactose and thiomannose are attached onto the double bonds of the PAGE‐blocks. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015 , 53, 2163–2174     

Combination of SDS‐PAGE and intact mass analysis for rapid determination of heterogeneities in monoclonal antibody therapeutics

mAbs are currently mainstream in biopharmaceuticals, and their market has been growing due to their high target specificity. Characterization of heterogeneities in mAbs is performed to secure their quality and safety by physicochemical analyses. However, they require time‐consuming task, which often strain the resources of drug development in pharmaceuticals. Rapid and direct method to determine the heterogeneities should be a powerful tool for pharmaceutical analysis. Considering the advantages of electrophoresis and MS, this study addresses the combination of SDS‐PAGE and intact mass analysis, which provides direct, rapid, and orthogonal determination of heterogeneities in mAb therapeutics. mAb therapeutics that migrated in SDS‐PAGE were recovered from gel by treatment with SDC‐containing buffer. Usage of SDC‐containing buffer as extraction solvent and ethanol‐based staining solution enhanced the recovery of intact IgG from SDS‐PAGE gels. Recovery of mAbs reached more than 86% with 0.2% SD. The heterogeneities, especially N ‐glycan variants in the recovered mAb therapeutics, were clearly determined by intact mass analysis. We believe that the study is important in pharmaceuticals‧ perspective since orthogonal combination of gel electrophoresis and intact mass analysis should be pivotal role for rapid and precise characterization of mAbs.     

Specific glutamic acid residues in targeted proteins induce exaggerated retardations in Phos‐tag SDS‐PAGE migration

We describe two unique proteins, Escherichia coli ClpX and human histone H2A, that show extremely retarded migrations relative to their molecular weights in Phos‐tag SDS‐PAGE, despite being nonphosphorylated. Although ClpX separated into multiple migration bands in Phos‐tag gels, the separation was not due to phosphorylation. The N‐terminal 47–61 region of ClpX was responsible for producing multiple phosphorylation‐independent structural variants, even under denaturing conditions, and some of these variants were detected as highly up‐shifted bands. By systematic Ala‐scanning mutation analysis in the N‐47–61 region, we concluded that the Glu‐51 or Glu‐54 residue was responsible for the appearance of exaggerated mobility‐shifting bands. Histone H2A showed a much slower migration in Phos‐tag gels in comparison with other major histones having similar molecular weights, and we found that the Glu‐62 or Glu‐65 residue caused the retarded migration. In addition, Phos‐tag SDS‐PAGE permitted us to detect a shift in the mobility of the phosphorylated form of histone H2A from that of the nonphosphorylated one. This is the first report showing that exaggerated retardation in the migration of a certain protein in Phos‐tag SDS‐PAGE is induced by interactions between the Phos‐tag molecule and the carboxylate group of a specific Glu residue on the target.     

A comparative analysis of human plasma and serum proteins by combining native PAGE,whole‐gel slicing and quantitative LC‐MS/MS: Utilizing native MS‐electropherograms in proteomic analysis for discovering structure and interaction‐correlated differences

MS identification has long been used for PAGE‐separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole‐gel slicing and quantitative LC‐MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty‐five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in‐gel digestion and quantitative LC‐MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1–28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as “native MS‐electropherograms”. Comparison of the electropherograms revealed significant plasma‐versus‐serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions.     

Native PAGE to study the interaction between the oncosuppressor p53 and its protein ligands

In the present study, we investigated a new approach for studying the interaction between p53 and MDM2/X (where MDM is murine double minute protein). The method is based on the different mobility between the interacting domains of the oncosuppressor p53 and its protein ligands MDM2/X on polyacrylamide gels under native conditions. While the two proteins MDM2/X alone were able to enter the gel, the formation of a binary complex between p53 and MDM2/X prevented the gel entry. The novel technique is reliable for determining the different affinity elicited by MDM2 or MDMX toward p53, and can be useful for analyzing the dissociation power exerted by other molecules on the p53–MDM2/X complex.     

A rapid and simple 8‐quinolinol‐based fluorescent stain of phosphoproteins in polyacrylamide gel after electrophoresis

In order to obtain an easy and rapid protocol to visualize phosphoproteins in SDS‐PAGE, a fluorescent detection method named 8‐Quinolinol (8‐Q) stain is described. 8‐Q can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion (Al³⁺) to the phosphate groups on the proteins and the metal chelating property of 8‐Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4～8 ng of α‐casein and β‐casein, 16～32 ng of ovalbumin and κ‐casein which is more sensitive than Stains‐All but less sensitive than Pro‐Q Diamond. The protocol of 8‐Q requires only 70 min in 0.75 mm mini‐size or 1.0 mm large‐size gels with five changes of solutions without destaining step; Pro‐Q takes at least 250 min with 11 changes of solutions. In addition, the new method was confirmed by the study of dephosphorylation and LC‐MS/MS, respectively. The approach to visualize phosphoprotein utilizing 8‐Q could be an alternative to simplify the analytical operations for phosphoproteomics research.     

Alternative visualization of SDS‐PAGE separated phosphoproteins by alizarin red S‐aluminum (III)‐appended complex

A novel fluorescence detection system using a chemosensor for phosphoprotein in gel electrophoretic analysis has been developed. The system employed the alizarin red S‐aluminum (III)‐appended complex as a fluorescent staining dye to perform the convenient and selective detection of phosphorylated proteins and total proteins in SDS‐PAGE, respectively. Therefore, a full and selective map of proteins can be achieved in the same process without resorting to other compatible detection methods. As low as 62.5 ng of α‐ (seven or eight phosphates) and β‐casein (five phosphates), 125 ng of ovalbumin (two phosphates), and κ‐casein (one phosphate) can be detected in approximately 135 min, with the linear responses of rigorous quantitation of changes over a 125–4000 ng range. As a result, alizarin red S‐aluminum (III) stain may provide a new choice for selective, economic, and convenient visualization of phosphoproteins.     

Design and synthesis of new fluorescent probe for rapid and highly sensitive detection of proteins via electrophoretic gel stain

A new fluorescent molecular probe, 2,2′‐(1E,1′E)‐2,2′‐(4‐(dicyanomethylene)‐4H‐pyrane‐2,6‐diyl)bis(ethene‐2,1‐diyl)bis(sodium benzenesulfonate) salt ( 1 ), possessing the cyanopyranyl moieties and two benzene sulfonic acid groups was designed and synthesized to detect proteins in solution and for high‐throughput SDS‐PAGE. Compound 1 exhibited no fluorescence in the absence of proteins; however, it exhibited strong fluorescence on the addition of bovine serum albumin as a result of intramolecular charge transfer. Compared with the conventional protocols for in‐gel protein staining, such as SYPRO Ruby and silver staining, 1 achieves higher sensitivity, even though it offers a simplified, higher throughput protocol. In fact, the total time required for protein staining was 60–90 min under optimum conditions much shorter than that required by the less‐sensitive silver staining or SYPRO Ruby staining protocols. Moreover, 1 was successfully applied to protein identification by mass spectrometry via in‐gel tryptic digestion, Western blotting, and native PAGE together with protein staining by 1 , which is a modified protocol of blue native PAGE (BN‐PAGE). Thus, 1 may facilitate high‐sensitivity protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.     

X-ray absorption fine structure of artificial antigens for cadmium

Immunoassay technology as a quick and large-scale screening method to detect metal ions in foods and environmental samples has rapidly been developed due to several advantages over conventional instrument-intensive methods. Unlike biomacromolecule, metal ions are haptens without immunogenicity, so successful preparation of artificial antigens is the first critical step for establishing immunoassay methods for them. In the current paper, cadmium ions were conjugated to BSA and OVA, respectively, using bifunctional chelator, p-SCN-Bn-DTPA. The ultraviolet analysis indicated that the maximum absorption peak of Cd–p-SCN-DTPA–BSA and Cd–p-SCN-DTPA–OVA had a small peak shift and an apparent absorbance increase compared to that of BSA and OVA, and the extents of substitution of ?-amino in both conjugates were 51.2% and 58.6%, respectively. In addition, the EXAFS of conjugates implied that Cd²⁺ coordinated with N and O atoms of DTPA in artificial antigens, the coordination type and number of Cd–DTPA, Cd–p-SCN-Bn-DTPA–BSA, Cd–p-SCN-Bn-DTPA–OVA were the same. XANES region and geometries of the three compounds were also same. These results implied that the three antigens had the similar local structure and atomic geometry.This was the first time that the XAFS was attempted for the identification of artificial heavy metal ion antigens.     

一种鉴定胸腺素高活性小分子组分的灵敏简便方法

用笔者稍加改进的梯度ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ尿素系统，分别测定自制的胸腺素Ｔ１溶液，观察到一条５．３ｋｕ的电泳谱带．Ｔ１溶液用Ｅ－玫瑰花结法测定时，生物活力较高，是粗抽提溶液的１８１倍