Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry.

Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide.

Magnetic nanoparticles of nitrogen enriched carbon (mnNEC) for analysis of pesticides and metabolites in zebrafish by gas chromatography-mass spectrometry.

Nanosized carbon based sorbents have been widely used for separation, enrichment and desalting of biological samples because of their distinguished characteristics. In this work, magnetic nanoparticles of nitrogen enriched carbon (mnNEC) have been developed for enrichment of organochlorine pesticide DDT and metabolite DDE that have been accumulated in zebrafish during the course of environmental exposure. Polymerization of pyrrole was performed in the aqueous suspension of Fe(3)O(4) nanoparticles. Resultant core-shell nanoparticules coated with polypyrrole were then subjected to a process of carbonization under high temperature and nitrogen atmospheric condition. The presence of nitrogen atoms in carbon nanoparticles increases the hydrophilicity and dispersability in aqueous samples. It has been experimentally demonstrated that mnNEC can be effectively dispersed in aqueous samples and rapidly isolated by the application of an external magnetic field. Recoveries of DDT and DDE from water range from 90% to 102% and 85-97% respectively. In combination with Selected Ion Monitoring (SIM) experiments of gas chromatography-mass spectrometry, the detection limit can be down to low ng/mL level. By using mnNEC approach, two interesting results have been found for zebrafish with 60 days exposure to DDT (1 µg/l). (1) There is higher concentration of DDT (37-143 ng/g) and DDE (173-1108 ng/g) in male zebrafish body tissues than that of female (7-52 ng/g and 146-362 ng/g for DDT and DDE respectively). (2) There is high ratio of DDE/DDT for both female and male zebrafish, implying high environmental persistence and ongoing bioaccumulation.

Desalting of phosphopeptides by tandem polypyrrole-c18 reverse phase micropipette tip (TMTip(PPY-C18)) based on hybrid electrostatic, Π-Π stacking and hydrophobic interactions for mass spectrometric analysis.

Desalting and concentration of peptides using reverse phase (RP) C18 chromatographic material based on hydrophobic interaction is a routine approach used in mass spectrometry (MS)-based proteomics. However, MS detection of small hydrophilic peptides, in particular, phosphopeptides that bear multiple negative charges, is challenging due to the insufficient binding to C18 stationary phase. We described here the development of a new desalting method that takes the unique properties of polypyrrole (PPY). The presence of positively charged nitrogen atoms under acidic conditions and polyunsaturated bonds in polypyrrole provide a prospect for enhanced adsorption of phosphopeptides or hydrophilic peptides through extra electrostatic and Π-Π stacking interactions in addition to hydrophobic interactions. In tandem with reversed phase C18 chromatographic material, the new type of desalting method termed as TMTip(PPY-C18) can significantly improve the MS detection of phosphopeptides with multiple phosphate groups and other small hydrophilic peptides. It has been applied to not only tryptic digest of model proteins but also the analysis of complex lysates of zebrafish eggs. The number of detected phosphate groups on a peptide ranged from 1 to 6. Particularly, polypyrrole based method can also be used in basic condition. Thus it provides a useful means to handle peptides that may not be detectable in acidic condition. It can be envisioned that the TMTip(PPY-C18) should be able to facilitate the exploration of large scale phosphoproteome.