• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文会议>ASMS Conference on Mass Spectrometry and Allied Topics >Global Proteomic Analysis of Stage I Endometrial Cancer Tissue to Evaluate Protein Expression in Low-and High-Risk Metastases
【24h】

Global Proteomic Analysis of Stage I Endometrial Cancer Tissue to Evaluate Protein Expression in Low-and High-Risk Metastases

机译：阶段I子宫内膜癌组织的全局蛋白质组学分析评价低和高风险转移中蛋白质表达

获取原文
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Endometrial cancer is the most common gynecologic-related cancer in women, responsible for the second highest gynecologic-cancer related patient mortality. While most endometrial cancers are confined to the uterus at diagnosis, approximately 15percent of apparent early stage cases have occult metastases and, therefore, destined to recur. Identification of molecular profiles associated with lymph node metastasis in early stage primary endometrial cancer specimens would be useful in directing future investigations towards preoperative identification of patients at high risk for metastasis. We have conducted a comprehensive proteomic analysis using nanoflow liquid chromatography coupled online to an Orbitrap mass spectrometer of laser-capture microdissected (LCM) endometrial cancer biopsies from over 100 individual patients to identify differentially expressed proteins associated with lymph node metastasis. Fresh frozen tumor tissue sections were collected by LCM in 0.2 mM 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-solfonate (PPS Silent~(TM) Surfactant, Protein Discovery, Knoxville, TN), a novel acid cleavable surfactant, containing 50 mM NaF, 0.1 mM Na_(3)VO_(4), 1 mM DTT and lysed by boiling for 10 min. Protein extracts were digested overnight (16 hours) with trypsin at a 1:50 ratio of trypsin:protein by weight. Following digestion, the PPS detergent was cleaved by lowering the pH of the solution by addition of an equivalent volume of neat formic acid and samples were incubated for 2 h at 30 deg C. The digests were lyophilized to dryness, resuspended in 0.1percent formic acid, and analyzed in duplicate by nanoflow reversed-phase liquid chromatography (nanoRPLC) coupled online via electrospray ionization to a hybrid linear ion trap-Orbitrap mass spectrometer (Thermo Fisher Scientific). The instrument was configured to collect a high resolution, high mass measurement accuracy broadband mass spectrum from which the seven-most abundant peptide molecular ions were dynamically selected for collision-induced dissociation (CID). Each data file represents approximately 30,000 CID spectra that were all searched using SEQUEST on a 72 processor Beowulf cluster against a UNIPROT derived human proteome database downloaded from the European Bioinformatics Institute (EBI). Only tryptic peptides with up to two missed cleavage sites meeting a specific SEQUEST scoring criteria [delta correlation ((DELTA)C_(n)) >= 0.08 and charge state dependent cross correlation (X_(corr)) >= 1.9 for [M+H]~(1+), >=2.2 for [M+2H]~(2+) and >=3.1 for [M+3H]~(3+)] were considered as legitimate identifications.
机译：子宫内膜癌是女性最常见的妇科相关癌症，负责第二高的妇科癌症病人相关的死亡率。虽然大多数子宫内膜癌被局限于子宫在诊断，大约15percent的表观早期病例有隐匿性转移，因此注定复发。与早期原发性子宫内膜癌样本淋巴结转移相关的分​​子概况的鉴定将在朝向患者术前识别在用于转移的高风险引导将来的调查是有用的。我们已经使用在线连接到显微切割从100个别患者（LCM）子宫内膜癌活检以识别与淋巴结转移相关联的差异表达的蛋白质激光捕获的轨道阱质谱仪纳流液相色谱法进行了全面的蛋白质组学分析。新鲜冷冻的肿瘤组织切片通过LCM在0.2mM的3- [3-（1,1-二bisalkyloxyethyl）吡啶-1-基]丙烷-1- solfonate（PPS无声〜（TM）表面活性剂，蛋白质的发现，诺克斯维尔，TN收集），一种新型酸可裂解的表面活性剂，含有50mM氟化钠，0.1mM的Na_（3）VO_（4），1mM DTT和通过10分钟煮沸裂解。重量的蛋白质：蛋白质萃取物用在1:50比率的胰蛋白酶胰蛋白酶消化过夜（16小时）。消化后，将PPS洗涤剂通过加入纯甲酸和样品的等效体积的降低溶液的pH值在30℃下温育2小时。将消化物冻干至干燥，再悬浮于0.1percent甲酸裂解，并在通过纳流式两份进行分析反相液相色谱（nanoRPLC）通过电喷雾离子化的在线耦合到线性离子阱，轨道阱质谱仪杂交体（赛默飞世尔科技）。该仪器被配置为收集高的分辨率，高的质量的测量精度宽带质谱从中七最丰富的肽分子离子动态地选择用于碰撞诱导解离（CID）。每个数据文件表示都使用针对一个UNIPROT 72处理器Beowulf集群上SEQUEST搜索衍生自欧洲生物信息研究所（EBI）下载人类蛋白质组数据库大约30,000 CID谱。仅胰蛋白酶肽具有高达满足特定SEQUEST评分标准[增量相关（（DELTA）C_（N））> = 0.08和充电状态相关的互相关（X_（更正））> = 1.9 [M + 2个错过切割位点H]〜（1+），> = 2.2 [M + 2H]〜（2+）和> = 3.1 [M + 3H]〜（3+）]被认为是合法的标识。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号