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ABSTRACT: A chemical proteomic approach was developed for profiling the noncovalent interactome of isoprenoid chain in the yeast proteome. A chemical probe that harbors a biotin moiety and a photoreactive benzophenone group linked to the terminal of geranyl group was synthesized. Photoaffinity labeling was performed by incubating the Saccharomyces cerevisiae proteome and the probe under 365 nm UV light. Thirty proteins were identified by immobilized NeutraAvidin enrichment, on-bead digestion, online 2-D nano-LC/MS/MS identification and semi-quantitative proteomic analysis. As noted by Gene Ontology annotation, the identified proteins demonstrate a wide range of catalytic activity in several biological processes, especially in metabolism and biosynthesis. Further data analysis shows that hydrophobic binding of the synthetic probe is potentially the major interaction force leading to covalent labeling. These results argue that intracellular allosteric interactions conferred by the isoprenoid chain of the corresponding chemical structures may be widespread at an interactomic level.
Proteomics 07/2008; 8(15):3094 - 3104. · 4.43 Impact Factor
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ABSTRACT: Enantiomeric separations in CEC with the macrocyclic antibiotic vancomycin immobilized silica monolith as a chiral stationary phase are presented. The monolithic silica capillary columns were prepared by a sol–gel process in fused-silica capillaries with an inner diameter of 50 μm and subsequently in situ immobilization of vancomycin as a chiral selector by reductive amination. Enantioselectivity was obtained for eight pairs of enantiomers in nonaqueous polar organic or aqueous mobile phases and most of them were baseline-separated with high column efficiencies. It was observed that the organic modifier ratio (MeOH/ACN) in the polar organic mobile phase played a significant role in controlling the resolution and efficiency of the enantiomers. In enantiomeric separation of propranolol, repeatability for column efficiency and resolution in the nonaqueous mobile phase was given in terms of RSD values at 1.1 and 2.3% (n = 5) for run-to-run injections and 7.2 and 9.6% (n = 5) for column-to-column testing while repeatability for the separation of thalidomide in the aqueous mobile phase was given in terms of RSD values at 1.5, 2.8% and 6.1, 10.5%, respectively.
Electrophoresis 07/2007; 28(15):2606 - 2612. · 3.30 Impact Factor
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ABSTRACT: Large-scale characterization of phosphoproteins requires highly specific methods for the purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed. Femtomole phosphopeptides from digestion products could be enriched by ZrO2 nanoparticles and can be well detected by MALDI mass spectrometric analysis. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano-LC-MS/MS) and MS/MS/MS. A total of 248 defining phosphorylation sites and 140 phosphorylated peptides were identified by manual validation using a series of rigid criteria.
Electrophoresis 06/2007; 28(13):2201 - 2215. · 3.30 Impact Factor
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ABSTRACT: An approach was developed to automate sample introduction for nanoflow LC-MS/MS (μLC-MS/MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 μm id×2 cm SCX trap column and a 75 μm id×12 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two-dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC-7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current μLC-MS/MS system. This system represented a promising platform for routine proteome analysis.
Proteomics 01/2007; 7(4):528 - 539. · 4.43 Impact Factor
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ABSTRACT: This review summarizes most of the recent developments in the preparation and application of polar stationary phases for CEC covering the literature published since the year 2004. These polar stationary phases have been adopted for separation of analytes by the modes of packing column CEC, open-tubular CEC (o-CEC) and monolithic column CEC. Currently, development of o-CEC using biomolecules, such as protein and DNA, as the immobilized ligands is highlighted partly due to the simplicity of preparation. Furthermore, monolithic columns have been extended quickly, particularly inorganic materials-based monoliths, such as silica, zirconia, hafnium, etc., as an alternative to packed columns have been developed quickly.
Electrophoresis 12/2006; 28(1‐2):148 - 163. · 3.30 Impact Factor
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ABSTRACT: A hybrid silica monolithic stationary phase for RP CEC was prepared by in situ co-condensation of (3-mercaptopropyl)-trimethoxysilane (MPTMS), phenyltriethoxysilane (PTES), and tetraethoxysilane (TEOS) via a sol–gel process. The thiol groups on the surface of the stationary phase were oxidized to sulfonic acids by peroxytrifluoroacetic acid. The introduced sulfonic acid moieties on the monoliths were characterized by a strong and relatively stable EOF in a broad pH range from 2.35 to 7.0 in CEC. Aromatic acids and neutral compounds can be simultaneously separated in this column under cathodic EOF. The CEC column exhibited a typical RP chromatographic mechanism for neutral compounds due to the introduced phenyl groups.
Electrophoresis 10/2006; 27(21):4266 - 4272. · 3.30 Impact Factor
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ABSTRACT: Monolithic silica capillary columns were prepared by a sol-gel process in fused-silica capillaries with an inner diameter of 50 μm and were modified by coating of cellulose tris(3,5-dimethylphenylcarbamate). Influences of the factors in the modification process on enantiomer separations were investigated. The prepared columns were used to perform enantiomer separations by CEC. Fifteen and two pairs of enantiomers were separated under aqueous and nonaqueous mobile phases, respectively, and most of them were baseline-separated with very high column efficiencies. The Van Deemter curve was found flat under high linear velocity of the mobile phase, which indicated favorable kinetic properties of the prepared columns. Baseline separation of a pair of enantiomers was achieved in 90 s with high-column efficiency by short-end separation under high voltage.
Electrophoresis 02/2006; 27(5‐6):1050 - 1059. · 3.30 Impact Factor
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ABSTRACT: A chemically bonded cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase (CSP) was prepared by a radical polymerization reaction. The prepared CSP was packed into fused-silica capillaries with inner diameter of 75 μm to perform enantiomer separations in CEC. The electrochromatographic behavior of the CSP was investigated. On the prepared CSP, high EOF could be generated under acidic mobile phases, which represented an advantage for the separation of acidic enantiomers. Several neutral, acidic, and basic enantiomers were resolved on the prepared CSP under aqueous mobile phases. The column efficiencies were between 20 000 and 100 000 plates/m, which were much higher than those of HPLC. In addition, it was observed that the separation of some enantiomers benefited from the adoption of THF as mobile phase modifier.
Electrophoresis 09/2005; 26(20):3921 - 3929. · 3.30 Impact Factor
