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Qi-Chang Xia

Publications and source records attributed to Qi-Chang Xia.

At least 19 recordsLinked to original sources
PubMed2003

Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing.

Leptospirosis is a widely spread disease of global concern. Infection causes flu-like episodes with frequent severe renal and hepatic damage, such as haemorrhage and jaundice. In more severe cases, massive pulmonary haemorrhages, including fatal sudden haemoptysis, can occur. Here we report the complete genomic sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroup Icterohaemorrhagiae consisting of a 4.33-megabase large chromosome and a 359-kilobase small chromosome, with a total of 4,768 predicted genes. In terms of the genetic determinants of physiological characteristics, the facultatively parasitic L. interrogans differs extensively from two other strictly parasitic pathogenic spirochaetes, Treponema pallidum and Borrelia burgdorferi, although similarities exist in the genes that govern their unique morphological features. A comprehensive analysis of the L. interrogans genes for chemotaxis/motility and lipopolysaccharide synthesis provides a basis for in-depth studies of virulence and pathogenesis. The discovery of a series of genes possibly related to adhesion, invasion and the haematological changes that characterize leptospirosis has provided clues about how an environmental organism might evolve into an important human pathogen.

Bacterial Adhesion↗
PubMed2003

Identification of proteins separated by one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis with matrix-assisted laser desorption/ionization ion trap mass spectrometry; comparison with matrix-assisted laser desorption/ionization time-of-flight mass fingerprinting.

Digests from ten gel bands containing low abundance proteins were analyzed by both matrix-assisted laser desorption/ionization ion trap (MALDI-IT) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) methods. MALDI-TOF techniques were able to identify only one protein from all 10 gel bands, while MALDI-IT identified eight proteins from the same 10 bands. The ability to perform MS/MS experiments with a MALDI-IT instrument leads to protein identifications based on both peptide molecular mass and sequence information, and is much less prone to errors and uncertainties introduced by peptide fingerprinting methodologies in which protein identification is based on peptide molecular masses alone.

Amino Acid Sequence↗
PubMed2002

Protein isoforms observed by ultrahigh resolution capillary isoelectric focusing electrospray ionization mass spectrometry.

On-line coupling of capillary isoelectric focusing (CIEF) to electrospray ionization mass spectrometry (ESI-MS) as a two-dimensional separation/analysis system was employed for high-resolution analysis of the protein isoforms observed during CIEF process. The analytical system was established by using neutral coated long capillary (80 cm), active capillary positioning and sheath-liquid interface. Proteins were separated and resolved in CIEF according to their differences in isoelectric point (pI), and then characterized by ESI-MS. The focused protein zones were eluted to the entrance of MS by combining cathodic mobilization with gravity. The ultrahigh resolution (difference in pI<0.04) of this technique obtained under certain conditions led to the detection of three isoforms in hemoglobin A and in sickle cell hemoglobin (with similar charge distribution and same molecular weight, but their differences in pIranging from 0.04 to 0.08) and two isoforms of beta-lactoglobulin A (difference in pI is 0.6). The isoelectric points, relative amounts, and molecular masses of these isoforms were determined simultaneously by CIEF-ESI-MS.

Carbonic Anhydrase II↗
PubMed2000

Synthesis of Human Proinsulin C-peptide and Preparation of Specific Antibody to It.

A HPLC and CE pure human proinsulin C-peptide was synthesized by solid-phase method and TSK column purification. Its amino acid sequence and MS were consistent with theoretical values. In comparison with the formly reported chemical synthesis of C-peptide, this method has the advantage of simplicity and higher overall yield (41%). To improve the immunogenicity and specificity of oligopeptide antibody, the acrylyl-C-peptides were transformed into a polymer the product had a poly-propionyl-core matrix with C-peptide branches. This treatment gave a macromolecule with a M(r) about 25 kD. By using the polymer to immunize New Zealand rabbits for 30 days, specific antiserum was obtained with titer of 2.5x10(4) (by ELISA), which did not cross react with BSA. Thus, the poly-propionyl-peptide system provided a new approach for preparing synthetic peptide antibody and therefore is promising for the preparation of synthetic peptide-based vaccine.

Journal Article↗
PubMed2000

Thiophosphorylation and Fluorescent Labeling of Substrate Peptide of Protein Kinase.

Study on the conditions of thiophosphorylation reaction and fluorescent labeling reaction of the substrate of protein kinase A was carried out by using Kemptide LRRASLG as a model. The suitable concentration of the fluorescent regent 5-{ ((2-iodoacetyl)amino)ethyl amino} naphthelene-1-sulfonic acid (1,5-IAEDANS) and the suitable pH of labeling reaction buffer were 1.6 mmol/L and 8, respectively. Stability of the labeled peptide under the conditions of automatic N-terminal protein sequencing, electrospray mass spectrometry and in the presence of 0.1% trifluoroacetic acid was investigated, respectively. The specific differences in UV spectra between the labeled and unlabeled peptides were observed. Therefore, the possibility to detect the thiophosphorylated and fluorophore labeled peptide during high performance liquid chromatography peptide mapping was primarily shown.

Journal Article↗
PubMed1999

Analysis of Peptide Mapping Glycosylated Site and Glycan Structure in Ribonuclease B by Liquid Chromatography-Electrospray Mass Spectrometry.

Liquid chromatography-electrospray mass spectrometry was utilized to analyze peptide mapping of a glycoprotein ribonuclease B to obtain its primary structure. The glycosylated site was determined by comparison of peptide mapping before and after glycanase treatment.Tandem MS(MS/MS)was performed to analyze the structure of N-linked glycan and deglycosylated peptide. The nature of glycan was determined to be of highmannose type by mass spectrometry after the treatment with alpha-mannosidase. In addition the relative abundance of heterogeneous glycopeptides was quantified. This method is rapid and sensitive for the characterization of glycoproteins with N-linked glycan.

Journal Article↗
PubMed1999

Application of Free-flow Electrophoresis to the Separation of Murine Lymphocytes.

Free-flow electrophoresis offers not only the all-in-solution separation process but also very gentle separation conditions, and it can be used for continuous separation and preparation, so it has been widely applied in the fields of biochemistry and cell biology. Using this technique to separate insoluble particles such as cells, both high separation efficiency and high activity and viability could be obtained. Murine lymphocytes were separated in low ionic strength triethanolamine buffer by free-flow electrophoresis, the cell fractions were detected and characterized by means of UV spectrometry, immune fluorescence labeling and flow cytometry. Results indicated that T and B lymphocytes were well separated with high viability.

Journal Article↗
PubMed1999

Protein and Peptide Identification by Capillary Zone Electrophoresis-Tandem Mass Spectrometry.

A method for highly sensitive protein and peptide identification by capillary zone electrophoresis/electrospray ionization-tandem mass spectrometry (CZE/ESI/MS/MS) was described. A mixture of peptides Met-enkephalin and Leu-enkephalin was analyzed, and their amino acid sequences were identified, respectively, using CZE/MS/MS. Peptide mapping for a tryptic digest of horse cytochrome c was performed by the same method. The results indicated that almost all peptides generated by tryptic digestion were identified, and their sequences were interpreted by their collision-induced (CID) mass spectra. Applying the uninterpreted CID spectra of the protein digest to searching in sequence database by the SEQUEST program could identify proteins conveniently and rapidly. The samples consumed in the CZE/MS/MS experiments were at picomole level, so this method is quite suitable for quality control of recombinant proteins and trace amount protein identification in the proteome research.

Journal Article↗
PubMed1999

Peptide Mapping and Primary Structure Analysis of hEGFby Mass Spectrometry.

Liquid chromatography-mass spectrometry(LC-MS) was adopted to analyze peptide mapping of the hEGF digested by TPCK-trypsin and V8 to determine the molecular weight of each peptide fragment. Most of the peptide fragments were sequenced by tandem MS(MS-MS) to obtain the amino acid sequence. In addition, the number of disulfide bonds was confirmed. This method will facilitate the determination and structural analysis for nature, synthesis and recombinant proteins and peptides. The accuracy and resolution are both better than that of the routine method.

Journal Article↗
PubMed1998

C-terminal Analysis of Proteins and Peptides.

A chemical method for C-terminal sequencing has been used to analyze the C-terminus of proteins and peptides, which can be converted to proteinyl alkylated thiohydantoin (proteinyl-ATH) or peptidyl-ATH via activated with acetic anhydride, coupled with [NCS](-) and alkylated with bromomethylene. The C-terminal ATH-amino acid can be cleaved and detected at 254 nm. The C-terminal sequences with varied lengths have been obtained from natural and recombinant proteins and peptides by the C-terminal analysis, and modifications and mutations of C-terminus have been found. By this method, important information of C-terminus of proteins and peptides can be obtained for the identification of recombinant products and sythetic peptides. At present, starting with samples of 1-2 nmole, C-terminal of 3-5 residues or that of over 10 residues in some case, can be successfully determined.

Journal Article↗
PubMed1998

Separation and Electrophoretic Behaviour of 8-aminonaphthalene-1,3,6-trisulfonic Acid-derivatized Oligosaccharides by Capillary Electrophoresis.

The mixture of the hydrolyzate of dextran was derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid(ANTS) by reductive amination, then the derivatives were separated by capillary zone electrophoresis in 50 mmol/L phosphate, pH 2.5 electrophoretic buffer and in the buffer of 100mmol/L disodium tetraborate, pH 9.3, respectively. Two-dimensional mapping of the series of linear dextranoligosaccharide derivatives was obtained by plotting their relative electrophoretic mobilities in acidic buffer vs those in alkaline buffer. The relative electrophoretic mobilities of the derivatives were linearly correlated to the negative two-thirds power of the molecular weights. The effects of triethylamine (TEA) added into the electrophoretic buffer on the electrophoretic behavior of the oligosaccharide derivatives were discussed. Those effects may arise from the interaction between the oligosaccharide derivatives and TEA. Similar results were found when we applied the methods above to mannan-, xylan- and chitin-oligosaccharide derivatives, respectively.

Journal Article↗
PubMed1998

Analysis of Monosaccharides Derivatized with 2-aminoacridone by Capillary Electrophoresis.

A new method for simultaneous analysis of 11 monosaccharides with reducing ends derivatized with 2-aminoacridone (AMAC) by the capillary electrophoresis in 300 mmol/L borate, pH 10.5 or 100 mmol/L disodium tetraborate and a pH 10.5 buffer was described. The chemical derivatization was performed at 45 degrees for 5 h, and then the derivatized monosaccharides were separated by capillary electrophoresis and detected at 264 nm. The chemical derivatization limit was 10 &mgr;mol/L. Concentration and mass detection limits of 0.6 &mgr;mol/L and 17 fmol, respectively, could be achieved. The linear correlation coefficients between the quotient of each integrated peak area divided by retention time and the concentration of monosaccharide were all greater than 0.992 (n=6) for the 11 monosaccharides in concentration ranging from 5 to 25 &mgr;mol/L. The relationship between the electrophoretic mobility of the derivatives and the structure of sugar moieties was discussed. The carbohydrate components of several glycoproteins were also by the method described above.

Journal Article↗
PubMed1998

Construction of Trichosanthin Mutant R122G and Its Activity Study.

AGG, the codon of 122 Arg of trichosanthin (TCS) was mutated to GGG (Gly) by U-DNA site-directed mutagenesis. The mutant TCS was expressed and purified from the supernatant of host cells, its activities were assayed and compared with expressed wild-type TCS. The results showed that the R122G TCS was still active as a RNA N-glycosidase but its ability to inhibit protein synthesis was 160-fold less than that of wild-type TCS, its abortifacient ability on mid-term gestated mice reduced from 100% to 9.3%, which suggested that 122 Arg was indirectly involved in the catalysis, and it played an important role in its bioactivities.

Journal Article↗
PubMed1998

Determination of the Binding Epitope for Anti-trichosanthin Monoclonal Antibody T(8)C(12).

Western blot result showed that T(8)C(12), an anti-trichosanthin (TCS) monoclonal antibody, could bind to a CNBr-cleaved TCS fragment with MW of 8 kD. The epitope was located in 1-72 of the N terminal of TCS as shown by amino acid analysis. A random 6-aa peptide library cloned in pIII of phage M13 was screened by T(8)C(12). After two cycles screening, 15 positive clones were randomly selected and six kinds of 6-aa sequences were determined, which showed to be highly homologous with a Ser/Thr-(X)-(X)-Arg motif, the X denoting hydrophobic amino acids. The motif was found to be similar to the first 3-5 amino acid sequence of the TCS N-terminal. The synthesized 1-8 peptide of TCS showed competitive binding to T(8)C(12) with native TCS. It suggested that 3-5 amino acid residues of TCS N-terminal was the core of epitope recognized by T(8)C(12).

Journal Article↗
PubMed1997

Non-radioactive Determination of Phosphorylation in Proteins or Peptides by Capillary Electrophoresis.

A new method of capillary electrophoresis was described which allowed the non-radioactive determination of all O-phosphoamino acids in peptides or proteins. It involved a partial hydrolysis of the peptide bonds, the derivatization of an amino acid mixture with phenylisothiocyanate and the separation of all the PTH-phosphoamino acids from other PTH-amino acids by capillary electrophoresis. The correlation coefficients of the linear least-squares regression curves of all the phophoamino acids in concentration ranging from 25 to 250 pmol/&mgr;l, were greater than 0.992 (n=6). Detection limits were in the fmol range. The method had been applied to the analysis of the phosphorylation sites in several model polypeptides and two nature phosphorylated proteins beta-casein and phosvitin.

Journal Article↗
PubMed1997

The Construction and Functional Study of Protein Kinase Inhibitor Phage.

A DNA fragment, which encoded the heat-stable protein kinase inhibitor (PKI) (5-24) of camp-dependent protein kinase (cAPK), was synthesized and cloned into phage display vector fe-tet-DOG1. Thus, PKI(5-24) was displayed on the surface of phage fd, which was termed PKI phage (cAPK inhibitor phage), in a form fused with gene III protein (g3p). It was showed that PKI phage not only repressed cAPK effectively, but also bound with the immobilized recombinant His(6)-tag mouse cAPK-Calpha( His(6)-mCalpha) specifically. The bound PKI phages could be quantitatively eluted under acidic conditions. Model affinity screening demonstrated that PKI phages could be selectively enriched from the mixture of PKI phage and wild-type phages (1:10(8)) using affinity chromatography of immobilized His(6)-mCalpha. These results suggest that selecting protein kinase inhibitor by phage display technique is feasible.

Journal Article↗