Using tissue engineering techniques for osteochondral repair and regeneration.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
X X Shao
Publications and source records attributed to X X Shao.
Capillary electrophoresis-electrospray mass spectrometry for the characterization of high-mannose-type N-glycosylation and differential oxidation in glycoproteins by charge reversal and protease/glycosidase digestion.
The characterization of high-mannose-type N-glycosylation by capillary electrophoresis-electrospray mass spectrometry (CE-ESI MS) was described. In addition to the use of a cationic noncovalent capillary coating, strong acidic buffer, and charge reversal to increase the glycoform resolving power, N-glycosidase F (PNGase F) combined with a basic protease and alpha-mannosidase combined with an acidic protease were used to analyze the high-mannose-type N-glycosylation in ribonuclease B (RNase B) and in a novel C-type lectin from the venom of Trimeresurus stejnegeri (TSL). The structures of oligosaccharide, glycosylation sites, and glycoform distributions were determined simultaneously, and the differential oxidation of Met residues in glycopeptides obtained from TSL protease digestion was also characterized successfully by CE-MS/MS. The results showed that the oligosaccharide attached to RNase B has a structure of GlcNAc2Man5 approximately 9, and that attached to TSL has a structure of GlcNAc2Min5 approximately 8. The glycoform distributions in these glycoproteins are quite different, with the GlcNAc2Man5 type predominant in RNase B, and the GlcNAc2Man8 type, in TSL This method may be useful not only for the characterization of glycosylation sites and glycan structures, but also for the determination of the relative abundance of individual glycoforms.
Alkylation↗
Proteome alterations in human hepatoma cells transfected with antisense epidermal growth factor receptor sequence.
The epidermal growth factor (EGF) is a member of the growth factor superfamily that can stimulate the proliferation of many types of cells. Overexpression of EGF receptor (EGFR) was observed in many types of cancer cells. Anti-EGFR antibodies or antisense nucleic acid sequences of EGFR can suppress the growth of hepatoma cells. In order to further investigate the proteome alterations associated with malignant growth of the human hepatoma cells and the influence of EGFR signal pathway on the cellular proteome, we have comparatively analyzed the proteomes of human hepatoma cells transfected with antisense EGFR sequence (cell strain JX-1) and its control cells (cell strain JX-0) by two-dimensional (2-D) gel electrophoresis and mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 40 protein spots showed significant expression changes in JX-1 cells compared to JX-0 cells. Three of them, including the tumor suppressor protein maspin, changed with tendency to the normal levels. Two protein spots were identified as HSP27 in the same gel, and one of them had a reduced level in JX-1 cells. The apparent alterations of HSP27 in expression level might be the results from their differential chemical modifications, suggesting the effect of dynamic post-translational modifications of proteins on the growth of hepatoma cells. Other proteins such as glutathione peroxidase (GPX-1) and 14-3-3-sigma also exhibited altered expression in JX-1 cells, and their functional implications are discussed.
Amino Acid Sequence↗
Determination of the disulfide bond pattern of a novel C-type lectin from snake venom by mass spectrometry.
The disulfide bond pattern of Trimeresurus stejnegeri lectin (TSL), a new member of the C-type lectin family, was determined by mass spectrometry. Four intrachain disulfide bonds of TSL, Cys(3)-Cys(14), Cys(31)-Cys(131), Cys(38)-Cys(133) and Cys(106)-Cys(123), and two interchain linkages, Cys(2)-Cys(2) and Cys(86)-Cys(86), were determined. Three strategies were used in this work. One intrachain (Cys(106)-Cys(123)) and one interchain (Cys(86)-Cys(86)) disulfide linkages were detected by standard MS methods. The disulfide bonds Cys(2)-Cys(2) and Cys(3)-Cys(14) were analyzed using a modified partial reduction procedure and MS/MS. The last two disulfide bonds were characterized by a MS/MS/MS technique. The strategies developed in this work could be applied more generally to detection of disulfide bond patterns.
Amino Acid Sequence↗
Identification of differentially expressed proteins between human hepatoma and normal liver cell lines by two-dimensional electrophoresis and liquid chromatography-ion trap mass spectrometry.
In the previous study, the proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 were separated by high resolution two-dimensional electrophoresis (2-DE). Image analysis revealed that 99 protein spots showed quantitative and qualitative variations that were significant (P < 0.01) and reproducible. Here we report the identification results of some of these protein spots. Protein spots excised from 2-D gels were subjected to in-gel digestion with trypsin, and the resulting peptides were measured by microbore high performance liquid chromatography - ion trap - mass spectrometry (LC-IT-MS) to obtain the tandem mass (MS/MS) spectra. Twelve protein spots were identified with high confidence using SEQUEST with uninterpreted MS/MS raw data. Besides inosine-5'-monophosphate dehydrogenase 2, heat shock 27 kDa protein, calreticulin and calmodulin, whose expression was elevated in hepatoma cells, glutathione-S-transferase P was identified from hepatoma cells in which its level was 18-fold higher compared to human liver cells. Two spots were identified as the homologs of reticulocalbin for the first time in hepatoma cells and their expression increased compared to liver cells. However, tubulin beta-1 chain and natural killer cell enhancing factor B were downregulated in hepatoma cells. A tumor suppressing serpin, maspin precursor, was identified from one spot whose quantity was much higher in the normal liver cell line. More interestingly, epidermal fatty acid-binding protein (E-FABP) and fatty acid-binding protein, adipocyte-type (A-FABP), were detected in liver cells but not in hepatoma cells. The functional implication of the identified proteins was discussed.
Chromatography, Liquid↗
Analysis of recombinant and modified proteins by capillary zone electrophoresis coupled with electrospray ionization tandem mass spectrometry.
A method for rapid characterization of recombinant and modified proteins with known sequences is described. The analytical system consists of a capillary zone electrophoresis (CZE) instrument coupled to an electrospray ionization ion trap tandem mass spectrometer via a sheath-flow interface. Following the procedure consists of proteolytic fragmentation, CZE peptide separation, tandem mass spectrometry (MS-MS) analysis of separated peptides, sequence database search and monitoring of the specific peptides, C 125 S mutated interleukin 2 (S-125-IL2) and bovine beta-casein were characterized as a model of recombinant protein and naturally modified protein, respectively. A tryptic peptide mixture derived from the synthetic salmon calcitonin (s-CT) was also analyzed to test the performance of the system. Although a conventional sheath-flow interface with much higher flow-rate compared to the microspray interface and nanospray interface was used, the proteins were identified at the low picomole level.
Amino Acid Sequence↗
Comparative study on the distribution of ovalbumin glycoforms by capillary electrophoresis.
Two commercial turkey egg ovalbumins (TEOs) with different quantities of mannose, were further purified by reversed-phase high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis for either of the purified glycoproteins showed one big wide band and one close small band. Capillary electrophoresis was used for the investigation of the separation of glycoforms of both glycoproteins. The best resolution of the glycoforms was obtained, reproducibly, with 100 mM borate, 1.8 mM 1,4-diaminobutane and pH 8.6 electrophoretic buffer. At least 13 glycoform peaks could be separated for either of the two glycoproteins. Their glycoform patterns were highly similar except for the conspicuous decrease in quantity of four glycoforms in the ovalbumin containing less mannose, compared to that of the other with more mannose. Coinjection electrophoresis of the two glycoproteins indicated that almost every glycoform peak of the former exactly overlapped with its corresponding glycoform peak of the latter. These results clearly indicated that the two TEOs possessed the same glycoform patterns but differed in quantity at least four glycoforms. It was found that the glycoform patterns were remarkably different between TEO and chicken egg ovalbumin.
Animals↗
Characterization of derivatization of sialic acid with 2-aminoacridone and determination of sialic acid content in glycoproteins by capillary electrophoresis and high performance liquid chromatography--ion trap mass spectrometry.
A simple and highly sensitive capillary electrophoresis (CE) method for determining the content of N-acetylneuraminic acid (Neu5Ac) in glycoproteins was developed. Neu5Ac was derivatized with 2-aminoacridone (AMAC) by reductive amination, and the AMAC-Neu5Ac adduct could be readily separated from the other 11 AMAC-derivatized neutral and acidic monosaccharides usually present in glycoproteins by CE in a 0.3 mol/L borate buffer, pH 10.5, and detected at 260 nm. The derivatization of Neu5Ac was achieved at 55 degrees C for 4 h. AMAC-Neu5Ac was stable at 20 degrees C in the dark for at least 12 h while at room temperature it spontaneously converted into another substance with a lower electrophoretic mobility, which was identified as decarboxylated AMAC-Neu5Ac by high performance liquid chromatography - ion trap mass spectrometry (HPLC-ITMS). Concentration and mass of Neu5Ac as low as 1 micromol/L and 35 fmol could be detected. The linear correlation coefficient between the ratio of peak area to migration time of AMAC-Neu5Ac and the concentration of Neu5Ac ranging from 10 to 120 micromol/L was 0.9978 (n=8). This method was successfully applied to the analysis of sialic acid in human urinary trypsin inhibitor (hu-UTI), bovine alpha1-acid glycoprotein (alpha1-AGP) and recombinant human erythropoietin (rhu-EPO). By combination of CE and HPLC-ITMS we found that N-glycolylneuraminic acid (Neu5Gc) was present in bovine alpha1-AGP in addition to Neu5Ac, with a quantity comparable to that of the latter.
Aminoacridines↗
Characterization and analysis of a novel glycoprotein from snake venom using liquid chromatography-electrospray mass spectrometry and Edman degradation.
An N-linked glycosylation in a novel C-lectin protein from snake venom was observed by Edman degradation and liquid chromatography-electrospray mass spectrometry. The peptides obtained by trypsin cleavage were analyzed to confirm the amino acid sequence and Asn5 was found to be the N-glycosylation site. The result was further confirmed by N-glycosidase digestion. In addition, the protein and tryptic peptides with and without glycan chain were characterized by mass spectrometry according to the mass difference. The glycopeptide obtained from proteolytic digestion was analyzed and the glycoforms were identified as high-mannose type by tandem MS coupled with alpha-mannosidase digestion. An oxidized Met residue was detected and located in the protein by mass spectrometry.
Amidohydrolases↗