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: 8168722
• 负责人: MING YOU
• 金额: $ 0.97万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-10 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168722
• 关键词: Acetonitriles; Albumins; Alkylation; Area; Binding Proteins; Bladder Neoplasm; Blood Proteins; CD44 Antigens; Cancer Model; Computer Retrieval of Information on Scientific Projects Database; Computer software; Control Animal; Cyclotrons; E-Cadherin; Fourier Transform; Funding; Grant; Hemoglobin; Hemopexin; Hepatic; High Pressure Liquid Chromatography; Hybrids; Individual; Institution; Ions; Isotopes; Label; Malignant neoplasm of urinary bladder; Mass Spectrum Analysis; Meprin; Methods; Nitrosamines; Palpable; Parents; Peptides; Phase; Proteins; Proteomics; Rattus; Research; Research Personnel; Resources; Retinoblastoma; Sampling; Scanning; Signal Transduction; Source; Trypsin; United States National Institutes of Health; Urine; comparative; 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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们使用无标记定量nanoESI-HPLC质谱方法对对照大鼠和带有4-羟丁基（丁基）亚硝胺诱导的膀胱肿瘤的大鼠的尿液进行比较蛋白质组学分析。在这项研究中，对照动物 (n = 10) 和可触及膀胱肿瘤的动物 (n = 10) 的尿蛋白在还原和烷基化后依次用内切蛋白酶 Lys C 和胰蛋白酶消化。使用纳流反相 HPLC 分离肽，使用纳喷雾电离进行电离，并使用混合线性四极傅里叶变换离子回旋质谱仪 (FT-MS) 进行分析。 20 个样品中每个样品的母体质量扫描均在 2 小时的乙腈线性梯度内获得。 将对照组和荷瘤组的质谱进行比对，并使用 Rosetta ElucidatorTM 软件对各个精确质量信号进行量化。两组之间具有统计显着差异面积（P < 0.01）的同位素组用于产生准确的 m/z 值，用于直接采集串联质谱以对肽进行测序并识别两组之间的蛋白质差异。在携带肿瘤的尿液中发现增加的蛋白质（在排除肝源性和公认的血液蛋白质，例如白蛋白、血红蛋白、血红蛋白之后）有meprin、E-钙粘蛋白、透明质酸受体、聚糖、视网膜母细胞瘤结合蛋白。