TARGETED, LABEL-FREE PROTEOMIC ANALYSIS OF URINE IN A RAT BLADDER CANCER MODEL

对大鼠膀胱癌模型中的尿液进行有针对性的、无标记的蛋白质组学分析

• 批准号: 8361368
• 负责人: MING YOU
• 金额: $ 0.61万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361368
• 关键词: Acetonitriles; Albumins; Alkylation; Area; Binding Proteins; Bladder Neoplasm; Blood Proteins; CD44 Antigens; Cancer Model; Computer software; Control Animal; Cyclotrons; E-Cadherin; Fourier Transform; Funding; Grant; Hemoglobin; Hemopexin; Hepatic; High Pressure Liquid Chromatography; Hybrids; Individual; Ions; Isotopes; Label; Malignant neoplasm of urinary bladder; Mass Spectrum Analysis; Meprin; Methods; National Center for Research Resources; Nitrosamines; Palpable; Parents; Peptides; Phase; Principal Investigator; Proteins; Proteomics; Rattus; Research; Research Infrastructure; Resources; Retinoblastoma; Sampling; Scanning; Signal Transduction; Source; Trypsin; United States National Institutes of Health; Urine; biomedical resource; comparative; cost; ionization; mass spectrometer; peptidyl-Lys metalloendopeptidase; protein aminoacid sequence; tumor; urinary

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We have used a label-free quantitative nanoESI-HPLC mass spectrometric method to perform comparative proteomics analysis on the urine from control rats and rats bearing 4-hydroxybutyl(butyl)nitrosamine induced bladder tumors. In this study the urinary proteins from control animals (n = 10) and from those with palpable bladder tumors (n = 10) were digested sequentially with endoprotease Lys C and trypsin after reduction and alkylation. The peptides were separated with nanoflow- reversed phase HPLC, ionized with nanospray ionization, and analyzed with a hybrid linear quadrupole Fourier transform ion cyclotron mass spectrometer (FT-MS). Parent mass scans were acquired over a 2 h linear gradient of acetonitrile for each of the 20 samples. The mass spectra from the control and tumor-bearing groups were aligned and the individual accurate mass signals were quantified using Rosetta ElucidatorTM software. The isotope groups with statistically significant different areas (P < 0.01) between the two groups were used to produce the accurate m/z values for directed acquisition of tandem mass spectra to sequence the peptide and identify the protein differences between the two groups. Among the proteins that were found to be increased in the tumor bearing urine (after excluding the hepatic-derived and well-recognized blood proteins e.g. albumin, hemopexin, hemoglobin) were meprin, E-cadherin, hyaluronan receptor, glycam, retinoblastoma binding protein.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 我们使用无标记定量 NanoESI-HPLC 质谱方法 对对照大鼠和妊娠大鼠的尿液进行比较蛋白质组学分析 4-羟基丁基（丁基）亚硝胺诱导的膀胱肿瘤。本研究中尿 来自对照动物 (n = 10) 和患有可触及膀胱肿瘤的动物 (n = 10)还原后依次用内切蛋白酶Lys C和胰蛋白酶消化 和烷基化。使用纳流反相 HPLC 分离肽， 通过纳喷雾电离进行电离，并使用混合线性四极傅里叶进行分析 变换离子回旋质谱仪（FT-MS）。 获得母体质量扫描 20 个样品均采用 2 小时乙腈线性梯度。 质谱 将对照组和荷瘤组的数据进行对齐，并且个体准确 使用Rosetta ElucidatorTM 软件对质量信号进行量化。同位素组 两组间面积差异有统计学意义（P < 0.01） 用于生成准确的 m/z 值，用于直接采集串联质量 光谱对肽进行测序并识别两者之间的蛋白质差异 组。在携带肿瘤的尿液中发现蛋白质增加 （排除肝源性和公认的血液蛋白（例如白蛋白）后， 血红蛋白、血红蛋白）分别为 meprin、E-钙粘蛋白、透明质酸受体、甘氨酸、 视网膜母细胞瘤结合蛋白。