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Wantao Ying

Publications and source records attributed to Wantao Ying.

11 recordsLinked to original sources
PubMed2006

A dataset of human fetal liver proteome identified by subcellular fractionation and multiple protein separation and identification technology.

A high throughput process including subcellular fractionation and multiple protein separation and identification technology allowed us to establish the protein expression profile of human fetal liver, which was composed of at least 2,495 distinct proteins and 568 non-isoform groups identified from 64,960 peptides and 24,454 distinct peptides. In addition to the basic protein identification mentioned above, the MS data were used for complementary identification and novel protein mining. By doing the analysis with integrated protein, expressed sequence tag, and genome datasets, 223 proteins and 15 peptides were complementarily identified with high quality MS/MS data.

Cell Membrane↗
PubMed2006

Proteome analysis of hepatocellular carcinoma by laser capture microdissection.

Hepatocellular carcinoma (HCC) is one of the most frequent visceral neoplasia worldwide and is a multifactorial and multistage pathogenesis that finally leads to the deregulation of cell homeostasis. Laser capture microdissection (LCM) may allow a more ready identification of differences in protein expression in selected cell types or areas of tissue, and microscopic regions as small as 3-5 microm in diameter can be sampled. Here we applied the LCM to the proteomic study of hepatitis B-related HCC and surrounding non-tumor tissues. Proteome alterations were observed using 2-DE and ESI-MS/MS, and alterations in the proteome were examined. Twenty protein spots were selected, of which 11 proteins were significantly altered in the HCC compared with the surrounding non-tumor tissues. Of the proteins that were selected, peroxiredoxin 2, apolipoprotein A-I precursor, 3-hydroxyacyl-CoA dehydrogenase type II, and 14.5-kDa translational inhibitor protein appear to be novel candidates as useful hepatitis B-related HCC markers. This study indicates that LCM is a useful technological method in the proteomic study of cancer tissue. The proteins revealed in this experiment can be used in the future for studies pertaining to hepatocarcinogenesis, or as diagnostic markers and therapeutic targets for HCC associated with hepatitis B virus infection.

Carcinoma, Hepatocellular↗
PubMed2006

Inflammation inhibitors were remarkably up-regulated in plasma of severe acute respiratory syndrome patients at progressive phase.

Severe acute respiratory syndrome (SARS) is a severe infectious disease that has affected many countries and regions since 2002. A novel member of the coronavirus, SARS-CoV, has been identified as the causative agent. However, the pathogenesis of SARS is still elusive. In this study, we used 2-D DIGE and MS to analyze the protein profiles of plasma from SARS patients, in the search for proteomic alterations associated with the disease progression, which could provide some clues to the pathogenesis. To enrich the low-abundance proteins in human plasma, two highly abundant proteins, albumin and IgG, were first removed. By comparing the plasma proteins of SARS patients with those of a normal control group, several proteins with a significant alteration were found. The up-regulated proteins were identified as alpha-1 acid glycoprotein, haptoglobin, alpha-1 anti-chymotrypsin and fetuin. The down-regulated proteins were apolipoprotein A-I, transferrin and transthyretin. Most of the proteins showed significant changes (up- or down-regulated) in the progressive phase of disease, and there was a trend back to normal level during the convalescent phase. Among these proteins, the alterations of fetuin and anti-chymotrypsin were further confirmed by Western blotting. Since all the up-regulated proteins identified above are well-known inflammation inhibitors, these results strongly suggest that the body starts inflammation inhibition to sustain the inflammatory response balance in the progression of SARS.

Adult↗
PubMed2006

Different immunoaffinity fractionation strategies to characterize the human plasma proteome.

Plasma proteins may often serve as indicators of disease and are a rich source for biomarker discovery. However, the intrinsic large dynamic range of plasma proteins makes the analysis very challenging because a large number of low abundance proteins are often masked by a few high abundance proteins. The use of prefractionation methods, such as depletion of higher abundance proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may ultimately prove to be informative biomarkers. But there are few studies on comprehensive investigation of the proteins both in the fractions depleted and remainder. In the present study, two different immunoaffinity fractionation columns for the top-6 or the top-12 proteins in plasma were investigated and both the proteins in column-bound and flow-through fractions were subsequently analyzed. A two-dimensional peptide separation strategy, utilizing chromatographic separation techniques, combined with tandem mass spectrometry (MS/MS) was employed for proteomic analysis of the four fractions. Using the established HUPO PPP criteria, a total of 2401 unique plasma proteins were identified. The Multiple Affinity Removal System yielded 921 and 725 unique proteins from the flow-through and bound fractions, respectively, whereas the Seppro MIXED 12 column yielded identification of 897 and 730 unique proteins from the flow-through and bound fractions, respectively. When more stringent criteria, based on searching against the reversed database, were implemented, 529 unique proteins were identified from the four fractions with the confidence in peptide identification increased from 73.6% to 99%. To determine whether the presence of nontarget proteins in the immunoaffinity-bound fraction could be attributed to their interaction with high abundance proteins, co-immunoprecipitation analysis with an antibody to human plasma albumin was performed, which resulted in an identification of 40 unique proteins from the coimmunoprecipitate with the more stringent criteria. This study illustrated that combining the column-bound and flow-through fractions from immunoaffinity separation affords more extensive profiling of the protein content of human plasma. The presence of nontarget proteins in the column-bound fractions may be induced by their binding to the higher abundance proteins targeted by the immunoaffinity column.

Amino Acid Sequence↗
PubMed2005

An approach to studying lung cancer-related proteins in human blood.

Early stage lung cancer detection is the first step toward successful clinical therapy and increased patient survival. Clinicians monitor cancer progression by profiling tumor cell proteins in the blood plasma of afflicted patients. Blood plasma, however, is a difficult cancer protein assessment medium because it is rich in albumins and heterogeneous protein species. We report herein a method to detect the proteins released into the circulatory system by tumor cells. Initially we analyzed the protein components in the conditioned medium (CM) of lung cancer primary cell or organ cultures and in the adjacent normal bronchus using one-dimensional PAGE and nano-ESI-MS/MS. We identified 299 proteins involved in key cellular process such as cell growth, organogenesis, and signal transduction. We selected 13 interesting proteins from this list and analyzed them in 628 blood plasma samples using ELISA. We detected 11 of these 13 proteins in the plasma of lung cancer patients and non-patient controls. Our results showed that plasma matrix metalloproteinase 1 levels were elevated significantly in late stage lung cancer patients and that the plasma levels of 14-3-3 sigma, beta, and eta in the lung cancer patients were significantly lower than those in the control subjects. To our knowledge, this is the first time that fascin, ezrin, CD98, annexin A4, 14-3-3 sigma, 14-3-3 beta, and 14-3-3 eta proteins have been detected in human plasma by ELISA. The preliminary results showed that a combination of CD98, fascin, polymeric immunoglobulin receptor/secretory component and 14-3-3 eta had a higher sensitivity and specificity than any single marker. In conclusion, we report a method to detect proteins released into blood by lung cancer. This pilot approach may lead to the identification of novel protein markers in blood and provide a new method of identifying tumor biomarker profiles for guiding both early detection and therapy of human cancer.

Adolescent↗
PubMed2005

Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome Project.

Based on the same HUPO reference specimen (C1-serum) with the six proteins of highest abundance depleted by immunoaffinity chromatography, we have compared five proteomics approaches, which were (1) intact protein fractionation by anion-exchange chromatography followed by 2-DE-MALDI-TOF-MS/MS for protein identification (2-DE strategy); (2) intact protein fractionation by 2-D HPLC followed by tryptic digestion of each fraction and microcapillary RP-HPLC/microESI-MS/MS identification (protein 2-D HPLC fractionation strategy); (3) protein digestion followed by automated online microcapillary 2-D HPLC (strong cation-exchange chromatography (SCX)-RPC) with IT microESI-MS/MS; (online shotgun strategy); (4) same as (3) with the SCX step performed offline (offline shotgun strategy) and (5) same as (4) with the SCX fractions reanalysed by optimised nanoRP-HPLC-nanoESI-MS/MS (offline shotgun-nanospray strategy). All five approaches yielded complementary sets of protein identifications. The total number of unique proteins identified by each of these five approaches was (1) 78, (2) 179, (3) 131, (4) 224 and (5) 330 respectively. In all, 560 unique proteins were identified. One hundred and sixty-five proteins were identified through two or more peptides, which could be considered a high-confidence identification. Only 37 proteins were identified by all five approaches. The 2-DE approach yielded more information on the pI-altered isoforms of some serum proteins and the relative abundance of identified proteins. The protein prefractionation strategy slightly improved the capacity to detect proteins of lower abundance. Optimising the separation at the peptide level and improving the detection sensitivity of ESI-MS/MS were more effective than fractionation of intact proteins in increasing the total number of proteins identified. Overall, electrophoresis and chromatography, coupled respectively with MALDI-TOF/TOF-MS and ESI-MS/MS, identified complementary sets of serum proteins.

Blood Proteins↗
PubMed2004

Proteomic analysis on structural proteins of Severe Acute Respiratory Syndrome coronavirus.

Recently, a new coronavirus was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with Severe Acute Respiratory Syndrome (SARS) and the causative association with SARS was determined. To reveal further the characteristics of the virus and to provide insight about the molecular mechanism of SARS etiology, a proteomic strategy was utilized to identify the structural proteins of SARS coronavirus (SARS-CoV) isolated from Vero E6 cells infected with the BJ-01 strain of the virus. At first, Western blotting with the convalescent sera from SARS patients demonstrated that there were various structural proteins of SARS-CoV in the cultured supernatant of virus infected-Vero E6 cells and that nucleocaspid (N) protein had a prominent immunogenicity to the convalescent sera from the patients with SARS, while the immune response of spike (S) protein probably binding with membrane (M) glycoprotein was much weaker. Then, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the complex protein constituents, and the strategy of continuous slicing from loading well to the bottom of the gels was utilized to search thoroughly the structural proteins of the virus. The proteins in sliced slots were trypsinized in-gel and identified by mass spectrometry. Three structural proteins named S, N and M proteins of SARS-CoV were uncovered with the sequence coverage of 38.9, 93.1 and 28.1% respectively. Glycosylation modification in S protein was also analyzed and four glycosylation sites were discovered by comparing the mass spectra before and after deglycosylation of the peptides with PNGase F digestion. Matrix-assisted laser desorption/ionization-mass spectrometry determination showed that relative molecular weight of intact N protein is 45 929 Da, which is very close to its theoretically calculated molecular weight 45 935 Da based on the amino acid sequence deduced from the genome with the first amino acid methionine at the N-terminus depleted and second, serine, acetylated, indicating that phosphorylation does not happen at all in the predicted phosphorylation sites within infected cells nor in virus particles. Intriguingly, a series of shorter isoforms of N protein was observed by SDS-PAGE and identified by mass spectrometry characterization. For further confirmation of this phenomenon and its related mechanism, recombinant N protein of SARS-CoV was cleaved in vitro by caspase-3 and -6 respectively. The results demonstrated that these shorter isoforms could be the products from cleavage of caspase-3 rather than that of caspase-6. Further, the relationship between the caspase cleavage and the viral infection to the host cell is discussed.

Amino Acid Sequence↗
PubMed2004

[Studies on relationship between peak capacity and column length for separation of peptides by high performance liquid chromatography].

In the study of proteomics, the high efficiency separation of peptides is very important for the identification of proteins and their interactions. Therefore, the relationship between peak capacity and column length for the separation of peptides on two reversed-phase columns with different lengths by reversed-phase high performance liquid chromatography was investigated and the effect of gradient times on peak capacity and peak width was examined. The experimental results showed that column length has significant effect on peak capacity. Furthermore, longer gradient times increase not only the peak number, but also the peak width, which is very favorable for the detection of more complicated mixture of peptides when they are separated and identified by capillary liquid chromatography combined with tandem mass spectrometry.

Chromatography, High Pressure Liquid↗
PubMed2003

Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis.

Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression.

Animals↗
PubMed2003

Proteome analysis on an early transformed human bronchial epithelial cell line, BEP2D, after alpha-particle irradiation.

To probe the mechanism of carcinogenesis of lung cancer at the molecular level and to find potential protein markers involved in the early phase of tumorgenesis, differential proteome analysis on primary passage cell line R15H, and early transformed cell line R15H20 derived from (238)Pu alpha-particle irradiation of human papillomavirus (HPV) 18-immortalized human bronchial epithelial cell line (BEP2D), was carried out using two-dimensional electrophoresis (2-DE) and peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Image analysis and Student's t-test (p < 0.05) showed that three protein spots were only expressed in R15H, intensities of 43 protein spots on the gels were altered between R15H and R15H20. Two of the three spots that were only expressed in R15H were identified as high mobility group protein 1. Two proteins decreased in abundance in R15H20 were identified as maspin precursor, a tumor suppressor and aminoacylase-1. Ornithine aminotransferase and peptidyl-prolyl cis-trans isomerase A that were increased in R15H20, were also identified. Relationships between these differentially expressed proteins and the carcinogenesis mechanism of lung cancer are discussed. The protein expression profile of the R15H cell line was also constructed during the study as a reference map for further comparative proteome analysis of the irradiation induced BEP2D cell line. Of the 90 spots analyzed with PMF in the 2-DE gel of R15H cell line, 50 proteins were identified by searching the nonredundant protein database SWISS-PROT/TrEMBL.

Alpha Particles↗