Xiao-Xia Shao

From Proteomic Analysis to Clinical Significance Overexpression of Cytokeratin 19 Correlates with Hepatocellular Carcinoma Metastasis

To better understand the mechanism underlying the hepatocellular carcinoma (HCC) metastasis and to search potential markers for HCC prognosis, differential proteomic analysis on two well-established HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/time-of-flight mass spectrometry. Cytokeratin 19 (CK19) was identified and found to be overexpressed in MHCC97-H as compared with MHCC97-L. This result was further confirmed by two-dimensional Western blot analysis and immunofluorescence assay. Furthermore, one-dimensional Western blot analysis showed consistently increased CK19 expression in progressively more metastatic cells. Immunohistochemical study on 102 human HCC specimens revealed that more patients in the CK19-positive group had overt intrahepatic metastases (satellite nodules, p < 0.05; vascular tumor emboli, p < 0.001; tumor node metastatis staging, p < 0.001). CK19 fragment CYFRA 21-1 levels measured in sera from nude mice model of human HCC metastasis with radioimmunoassay increased in parallel with tumor progression and rose remarkably when pulmonary metastases occurred. The results demonstrated that overexpression of CK19 in HCC cells is related to metastatic behavior. Serum CK19 level might reflect the pathological progression in some HCC and may be a useful marker for predicting tumor metastasis and a therapeutic target for the treatment of HCC patients with metastases.

Identification of differentially expressed proteins between human hepatoma and normal liver cell lines by two‐dimensional electrophoresis and liquidchromatography‐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‐transferse 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.

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.

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°C for 4 h. AMAC‐Neu5Ac was stable at 20°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 μmol/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 μmol/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 α1‐acid glycoprotein (α1‐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 α1‐AGP in addition to Neu5Ac, with a quantity comparable to that of the latter.

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.

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.

Identification of phosphorylation sites of proteins by high performance liquid chromatography-electrospray ionization-quadrupole ion trap mass spectrometry

The phosphorylation sites of two phosphorylated proteins, bovine β-casein and myelin basic protein (MBP), were identified by high performance liquid chromatography-electrospray ionization-quadrupole ion trap mass spectrometry (HPLC-ESI-QITMS). The tryptic digest of each protein was separated by HPLC, the molecular weight of each peptide was determined by ESI-QITMS on line, and MS/MS spectrum of each peptide was simultaneously obtained by the combination of collision-induced desorption (CID) technique and tandem mass spectrometry (MS/MS) of QITMS. The phosphorylated peptide was identified by looking into whether the difference between the observed and predicted molecular weights of a peptide is 80 u or its integral multiple. Then the phosphorylation site was identified through manual interpretation of the MS/MS spectrum of the phosphorylated peptide or automatic SEQUEST data base-searching.

Identification of two-dimensional electrophoresis-separated proteins in human hepatoma cell by electrospray ion trap mass spectrometry

As one of the most important analytical methods in proteome research, mass spectrometry was utilized to identify proteins separated by two-dimensional electrophoresis in the human hepatoma cell line BEL-7404. The protein spots were excised from the gel, followed by in-gel digestion, and the peptide mappings were analyzed by liquid chromatography electrospray ion trap mass spectrometer. Nine proteins were identified via database searching, according to the molecular weights and amino acid sequences of peptides, among which two proteins have not been identified in the other liver-cell database. The sequence coverage was 21% –72%. Furthermore, the relationship between the expressed proteins and the liver carcinoma was discussed.