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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 我们已经使用了无标记的定量纳米纳米 - 纳米级质谱法 对控制大鼠和轴承大鼠的尿液进行比较蛋白质组学分析 4-羟基丁酰基（丁基）亚硝胺诱导的膀胱肿瘤。在这项研究中 来自对照动物的蛋白质（n = 10）和那些患有明显膀胱肿瘤的蛋白质（n = 10）在还原后用内蛋白酶Lys C依次消化 和烷基化。用纳米流相反相HPLC分离肽， 用纳米喷雾电离电离，并用杂化线性四极傅里极傅立叶分析 转换离子回旋质谱仪（FT-MS）。 获得了家长群众扫描 在20个样品中的每一个中，乙腈的线性梯度超过2小时。 质谱 从对照组和承重组对齐，并且个体准确 使用Rosetta Elucidatortm软件对质量信号进行定量。同位素组 两组之间具有统计学意义的不同区域（p <0.01）是 用于产生准确的M/z值，以导向串联质量 光谱以对肽进行测序并确定两者之间的蛋白质差异 组。在发现尿液肿瘤中发现蛋白质增加的蛋白质中 （在排除肝衍生且公认的血蛋白之后，例如白蛋白， 血红蛋白，血红蛋白）是Meprin，E-钙黏着蛋白，透明质酸受体，聚糖， 视网膜母细胞瘤结合蛋白。