| Bladder cancer remains to be a focus in cancer research as it is the most prevalent type of cancer in the urinary tract, most of the cases of bladder cancer are transitional cell carcinoma (BTCC) . The majority of the bladder tumors (>70%) are diagnosed as superficial and easily treated by transurethraldissection and chemotherapy . But these tumors exhibit a high frequency of recurrence and some of them will progress to muscle-invasive and metastatic tumors and about 10 - 15% of them will progress to life-threatening malignancies . The pathological staging and grading system is currently the most significant factor for therapeutic interventions and clinical outcome. However, it is not possible to predict the recurrence and to understand bladder carcinogenesis by pathological classification. Therefore it is necessary to identify biomarkers that are useful in prognosis, progression, and clinical medicine.In order to improve the diagnosis and prognosis of BTCC, there is an urgent need to identify molecular markers to detect the disease. Usingtissue samples from patients with BTCC may be the most direct and persuasive way to find useful diagnostic and/or prognostic markers. Recently, proteomic analysis was applied to BTCC tissues.Because the carcinogenesis in BTCC involves multiple cellular changes. An improved understanding of the molecular mechanisms underlying carcinogenesis of BTCC is essential to develop novel and more effective molecular targets for therapy and identify patients with the highest risk for disease relapse. Recent advances in the fields of genomics and proteomics give rise to promise for the better understanding of the molecular events that are associated with tumor carcinogenesis as well as identifying molecular markers and therapeutic targets.Since the biological processes are directly executed by proteins which are dynamically modified and processed at multiple levels during or after their maturation, the state of an organism is essentially reflected in its proteoroe rather than genome, which is much more stable. An important step towards better understanding the carcinogenesis of bladder is to profile protein expression and to look for markers that are deregulated. To begin with, we want to established the proteome map and have found differential protein expression between high malignant BTCC and its normal epithelium samples. Protein identification by MS reveals alteration of expression in a group of proteins that have not been reported previously in bladder cancer.LC MS/MS was performed using an LTQ linear Itmass spectrometer (Thermo, San Jose, CA, USA). The system was fitted with a C18 KP column.Mobile phase A (0. 1% formic acid in water) and the mobile phase B (0. 1% formic acid in ACN) were selected. The tryptic peptide mixtures were eluted using a gradient of 2 â– â– 98% B over 180 min. The 1..TQ Linear ITmass .spectrometer was set so that one full MS scanwas followed by ten MS/MS scans on the ten most intense ions from the MS spectrum with the following Dynamic Exclusion? settings: repeat count 2, repeat duration 30 s, exclusion duration90 s. Each sample was analyzed in triplicate. To identify proteins in the above sample, spectra form each fraction was searched automatically with the the TurboSEQUEST program the BioWorks?D 3. 1 software suite against the nonredundant human protein database from IPL An accepted SEQUEST result had to have a Delta CN score of at. least 0. 1 (regardless of charge state). A singly charged peptide must be tryptic, and the crosscorrelation score (Xcorr) had to be at least 1.9. Tryptic or partially tryptic pep tides with a charge state of f-2 have an Xcorr of at least 2.2. Triply charged tryptic or partially tryptic peptides with a +3 charge state was accepted if the Xcorr was ^ 3. 75 . Generally, an Xcorr greater than 2. 0 indicates a highly significant match, and a DeltaCn higher than 0. 1 indicates a significant distinction between the best match and the second-best match. The pi and Mr of the proteins were analyzed using ExPASy proteomics tools accessed from en. expasy. org/tools/#proteome. The general average hydropathicity or GRAVY score is calculated as the arithmetic mean of the sum of the hydropathic indices of each amino acid . We conducted the transmembrane prediction using the program TMHMM 2. 0, which can be accessed from theCenter for Biological Sequence Analysis (www, ebs. dtu. dk/services /TMHMM/). All identified proteins were classified by their molecular function, cellular component, and biological process with the tools on www. geneontology. org.200 u g of solubilized proteins from BTCC tissues were reduced, alkylated, and digested with trypsin, and the resulting peptides were separated on a capillary C-18 column and then detected by ion trap mass spectrometry(lD~LC-MS/MS). A total of 508 and 542 proteins were unambiguously identified in tumor and normal tissue, respectively. By bioinformatic analysis, 53(10.4%), 65 (12.0%) hydrophobic proteins were found in high malignant BTCC and its normal epithelium sample, respectively. We conducted the transmembrane mapping prediction using the program TMHMM 2. 0 and found 55(10. 8%), and 61 (11. 3%) transmembrane proteins in high malignant BTCC and its normal epithelium sample, respectively. Among them, 27 and 31 proteins with one trans-membrane region, 4 ang 6 proteins with two trans-membrane regions, and 19 and 16 proteins with at least three trans-membrane region, respectively.The pi and Mr of the proteins were analyzed using ExPASy proteomics tools, our strategy appeared to give an overall understanding of proteins in high malignant BTCC with little restrictions on the Mw and pi, There were 110(21. 7%)and 127 (23.4%) proteins with Mr beyond routine 2DE (Mr <10 kDa, Mr MOO kDa);61 (12.0%)and 80 (14.8%) with pi beyond traditional 2DE (pi >9) in high malignant BTCC and its normal epithelium sample,respectively. Funtional analysis with GO tools , the amountproteins with participate cell proliferation , cell adhesion and antioxidation increasing obviously.Using proteomics approaches, we have profiled the protein expression of in high malignant BTCC and its normal epithelium sample and identified the differentially expressed proteins, such as PRDX6 > Aquaporin-1 . it. provid original path to discover effective marker. And it is necessary to validated with downstream experiments.
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